Death domain containing receptor-4 agonist antibodies and methods

ABSTRACT

The present invention relates to novel Death Domain Containing Receptor-4 (DR4) proteins which are members of the tumor necrosis factor (TNF) receptor family. In particular, isolated nucleic acid molecules are provided encoding the human DR4 proteins. DR4 polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of DR4 activity.

The present application is a divisional of U.S. patent application Ser.No. 09/448,868, filed Nov. 24, 1999, now U.S. Pat. No. 6,461,823, whichis a divisional of U.S. Pat. application Ser. No. 09/013,895, filed Jan.27, 1998, now U.S. Pat. No. 6,342,363, which claims the benefit of U.S.Provisional Application No. 60/037,829, filed Feb. 5, 1997 and U.S.Provisional Application No. 60/035,722 filed Jan. 28, 1997; which areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to a novel member of the tumor necrosisfactor family of receptors. More specifically, isolated nucleic acidmolecules are provided encoding human Death Domain Containing Receptor4, sometimes herein “DR4”. DR4 polypeptides are also provided, as arevectors, host cells and recombinant methods for producing the same. Theinvention further relates to screening methods for identifying agonistsand antagonists of DR4 activity.

BACKGROUND OF THE INVENTION

Many biological actions, for instance, response to certain stimuli andnatural biological processes, are controlled by factors, such ascytokines. Many cytokines act through receptors by engaging the receptorand producing an intra-cellular response.

For example, tumor necrosis factors (TNF) alpha and beta are cytokineswhich act through TNF receptors to regulate numerous biologicalprocesses, including protection against infection and induction of shockand inflammatory disease. The TNF molecules belong to the “TNF-ligand”superfamily, and act together with their receptors or counter-ligands,the “TNF-receptor” superfamily. So far, nine members of the TNF ligandsuperfamily have been identified and ten members of the TNF-receptorsuperfamily have been characterized.

Among the ligands there are included TNF-α, lymphotoxin-α (LT-α, alsoknown as TNF-β), LT-β (found in complex heterotrimer LT-α2-β), FasL,CD40L, CD27L, CD30L, 4–1BBL, OX40L and nerve growth factor (NGF). Thesuperfamily of TNF receptors includes the p55TNF receptor, p75TNFreceptor, TNF receptor-related protein, FAS antigen or APO-1, CD40,CD27, CD30, 4-1BB, OX40, low affinity p75 and NGF-receptor (Meager, A.,Biologicals, 22:291–295 (1994)).

Many members of the TNF-ligand superfamily are expressed by activatedT-cells, implying that they are necessary for T-cell interactions withother cell types which underlie cell ontogeny and functions. (Meager,A., supra).

Considerable insight into the essential functions of several members ofthe TNF receptor family has been gained from the identification andcreation of mutants that abolish the expression of these proteins. Forexample, naturally occurring mutations in the FAS antigen and its ligandcause lymphoproliferative disease (Watanabe-Fukunaga, R., et al., Nature356:314 (1992)), perhaps reflecting a failure of programmed cell death.Mutations of the CD40 ligand cause an X-linked immunodeficiency statecharacterized by high levels of immunoglobulin M and low levels ofimmunoglobulin G in plasma, indicating faulty T-cell-dependent B-cellactivation (Allen, R. C. et al., Science 259:990 (1993)). Targetedmutations of the low affinity nerve growth factor receptor cause adisorder characterized by faulty sensory innovation of peripheralstructures (Lee, K. F. et al., Cell 69:737 (1992)).

TNF and LT-α are capable of binding to two TNF receptors (the 55- and75-kd TNF receptors). A large number of biological effects elicited byTNF and LT-α, acting through their receptors, include hemorrhagicnecrosis of transplanted tumors, cytotoxicity, a role in endotoxicshock, inflammation, immunoregulation, proliferation and anti-viralresponses, as well as protection against the deleterious effects ofionizing radiation. TNF and LT-α are involved in the pathogenesis of awide range of diseases, including endotoxic shock, cerebral malaria,tumors, autoimmune disease, AIDS and graft-host rejection (Beutler, B.and Von Huffel, C., Science 264:667–668 (1994)). Mutations in the p55Receptor cause increased susceptibility to microbial infection.

Moreover, an about 80 amino acid domain near the C-terminus of TNFR1(p55) and Fas was reported as the “death domain,” which is responsiblefor transducing signals for programmed cell death (Tartaglia et al.,Cell 74:845 (1993)).

Apoptosis, or programmed cell death, is a physiologic process essentialto the normal development and homeostasis of multicellular organisms (H.Steller, Science 267, 1445–1449 (1995)). Derangements of apoptosiscontribute to the pathogenesis of several human diseases includingcancer, neurodegenerative disorders, and acquired immune deficiencysyndrome (C. B. Thompson, Science 267, 1456–1462 (1995)). Recently, muchattention has focused on the signal transduction and biological functionof two cell surface death receptors, Fas/APO-1 and TNFR-1 (J. L.Cleveland, et al., Cell 81, 479–482 (1995); A. Fraser, et al., Cell 85,781–784 (1996); S. Nagata, et al., Science 267, 1449–56 (1995)). Bothare members of the TNF receptor family which also include TNFR-2, lowaffinity NGFR, CD40, and CD30, among others (C. A. Smith, et al.,Science 248, 1019–23 (1990); M. Tewari, et al., in Modular Texts inMolecular and Cell Biology M. Purton, Heldin, Carl, Ed. (Chapman andHall, London, 1995). While family members are defined by the presence ofcysteine-rich repeats in their extracellular domains, Fas/APO-1 andTNFR-1 also share a region of intracellular homology, appropriatelydesignated the “death domain”, which is distantly related to theDrosophila suicide gene, reaper (P. Golstein, et al., Cell 81, 185–6(1995); K. White et al., Science 264, 677–83 (1994)). This shared deathdomain suggests that both receptors interact with a related set ofsignal transducing molecules that, until recently, remainedunidentified. Activation of Fas/APO-1 recruits the deathdomain-containing adapter molecule FADD/MORT1 (A. M. Chinnaiyan, et al.,Cell 81, 505–12 (1995); M. P. Boldin, et al., J. Biol Chem 270, 7795–8(1995); F. C. Kischkel, et al., EMBO 14, 5579–5588 (1995)), which inturn binds and presumably activates FLICE/MACH1, a member of theICE/CED-3 family of pro-apoptotic proteases (M. Muzio et al., Cell 85,817–827 (1996); M. P. Boldin, et al., Cell 85, 803–815 (1996)). Whilethe central role of Fas/APO-1 is to trigger cell death, TNFR-1 cansignal an array of diverse biological activities-many of which stem fromits ability to activate NF-kB (L. A. Tartaglia, et al., Immunol Today13, 151–3 (1992)). Accordingly, TNFR-1 recruits the multivalent adaptermolecule TRADD, which like FADD, also contains a death domain (H. Hsu,et al., Cell 81, 495–504 (1995); H. Hsu, et al., Cell 84, 299–308(1996)). Through its associations with a number of signaling moleculesincluding FADD, TRAF2, and RIP, TRADD can signal both apoptosis andNF-kB activation (H. Hsu, et al., Cell 84, 299–308 (1996); H. Hsu, etal., Immunity 4, 387–396 (1996)).

Recently a new apoptosis inducing ligand was discovered. Wiley, S. R. etal., refer to the new molecule as TNF-related apoptosis-inducing ligandor (“TRAIL”) (Immunity 3:673–682 (1995)). Pitti, R. M. et al., refer tothe new molecule as Apo-2 ligand or (“Apo-2L”). This molecule was alsodisclosed in copending U.S. Provisional Patent Application Ser. No.60/013405. For convenience, it will be referred to herein as TRAIL.

Unlike FAS ligand whose transcripts appear to be largely restricted tostimulated T-cells, significant levels of TRAIL are seen in manytissues, and it is constitutively transcribed by some cell lines. It hasbeen shown that TRAIL acts independently from FAS ligand (Wiley, S. R.,et al. (1995)), supra). Studies by Marsters, S. A. et al., haveindicated that TRAIL activates apoptosis rapidly, within a time framethat is similar to death signalling by FAS/Apo-1L but much faster thanTNF-induced apoptosis (Current Biology, 6:750–752 (1996)). All work todate suggest that the receptor for TRAIL is not one of the many knownTNF-receptors.

The effects of TNF family ligands and TNF family receptors are variedand influence numerous functions, both normal and abnormal, in thebiological processes of the mammalian system. There is a clear need,therefore, for identification and characterization of such receptors andligands that influence biological activity, both normally and in diseasestates. In particular, there is a need to isolate and characterize thereceptor for the newly discovered TRAIL ligand.

SUMMARY OF THE INVENTION

The present invention provides for isolated nucleic acid moleculescomprising nucleic acid sequences encoding the amino acid sequence shownin FIG. 1 (SEQ ID NO:2) or the amino acid sequence encoding the cDNAclone deposited as ATCC Deposit No. 97853 on Jan. 21, 1997.

The present invention also provides vectors and host cells forrecombinant expression of the nucleic acid molecules described herein,as well as to methods of making such vectors and host cells and forusing them for production of DR4 polypeptides or peptides by recombinanttechniques.

The invention further provides an isolated DR4 polypeptide having anamino acid sequence encoded by a polynucleotide described herein.

The present invention also provides diagnostic assays such asquantitative and diagnostic assays for detecting levels of DR4 protein.Thus, for instance, a diagnostic assay in accordance with the inventionfor detecting over-expression of DR4, or soluble form thereof, comparedto normal control tissue samples may be used to detect the presence oftumors.

Tumor Necrosis Factor (TNF) family ligands are known to be among themost pleiotropic cytokines, inducing a large number of cellularresponses, including cytotoxicity, anti-viral activity, immunoregulatoryactivities, and the transcriptional regulation of several genes.Cellular response to TNF-family ligands include not only normalphysiological responses, but also diseases associated with increasedapoptosis or the inhibition of apoptosis. Apoptosis-programmed celldeath-is a physiological mechanism involved in the deletion ofperipheral T lymphocytes of the immune system, and its dysregulation canlead to a number of different pathogenic processes. Diseases associatedwith increased cell survival, or the inhibition of apoptosis, includecancers, autoimmune disorders, viral infections, inflammation, graft v.host disease, acute graft rejection, and chronic graft rejection.Diseases associated with increased apoptosis include AIDS,neurodegenerative disorders, myelodysplastic syndromes, ischemic injury,toxin-induced liver disease, septic shock, cachexia and anorexia.

Thus, the invention further provides a method for enhancing apoptosisinduced by a TNF-family ligand, which involves administering to a cellwhich expresses the DR4 polypeptide an effective amount of an agonistcapable of increasing DR4 mediated signaling. Preferably, DR4 mediatedsignaling is increased to treat a disease wherein decreased apoptosis isexhibited.

In a further aspect, the present invention is directed to a method forinhibiting apoptosis induced by a TNF-family ligand, which involvesadministering to a cell which expresses the DR4 polypeptide an effectiveamount of an antagonist capable of decreasing DR4 mediated signaling.Preferably, DR4 mediated signaling is decreased to treat a diseasewherein increased apoptosis is exhibited.

Whether any candidate “agonist” or “antagonist” of the present inventioncan enhance or inhibit apoptosis can be determined using art-knownTNF-family ligand/receptor cellular response assays, including thosedescribed in more detail below. Thus, in a further aspect, a screeningmethod is provided for determining whether a candidate agonist orantagonist is capable of enhancing or inhibiting a cellular response toa TNF-family ligand. The method involves contacting cells which expressthe DR4 polypeptide with a candidate compound and a TNF-family ligand,assaying a cellular response, and comparing the cellular response to astandard cellular response, the standard being assayed when contact ismade with the ligand in absence of the candidate compound, whereby anincreased cellular response over the standard indicates that thecandidate compound is an agonist of the ligand/receptor signalingpathway and a decreased cellular response compared to the standardindicates that the candidate compound is an antagonist of theligand/receptor signaling pathway. By the invention, a cell expressingthe DR4 polypeptide can be contacted with either an endogenous orexogenously administered TNF-family ligand.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A–1C show the nucleotide sequence (SEQ ID NO:1) and deduced aminoacid sequence (SEQ ID NO:2) of DR4. It is predicted that amino acids1–23 constitute the signal peptide, amino acids 24–238 constitute theextracellular domain, amino acids 239–264 constitute the transmembranedomain, and amino acids 265–468 constitute the intracellular domain ofwhich amino acids 379–422 constitute the death domain.

FIGS. 2A–2C show the regions of similarity between the amino acidsequences of DR4, human tumor necrosis factor receptor 1 (SEQ ID NO:3),human Fas protein (SEQ ID NO:4), and the death domain containingreceptor 3 (DR3) (SEQ ID NO:5).

FIG. 3 shows an analysis of the DR4 amino acid sequence. Alpha, beta,turn and coil regions; hydrophilicity and hydrophobicity; amphipathicregions; flexible regions; antigenic index and surface probability areshown. In the “Antigenic Index—Jameson-Wolf” graph, amino acid residues35–92, 114–160, 169–240, 267–298, 330–364, 391–404, and 418–465 in FIG.1 correspond to the shown highly antigenic regions of the DR4 protein.

FIG. 4 shows the nucleotide sequences of related nucleic acid fragmentsHTOIY07R (SEQ ID NO:6) and HTXEY80R (SEQ ID NO:7).

FIGS. 5A and 5B show the ability of DR4 to induce apoptosis in the celllines MCF7 and 293. FIG. 5C shows the ability of death proteaseinhibitors z-VAD-fmk and CrmA to inhibit the apoptotic action of DR4.

FIG. 6A shows the ability of a soluble extracellular DR4-Fc fusion toblock the apoptotic inducing ability of TRAIL. FIG. 6B shows theinability of soluble extracellular DR4-Fc fusion to block the apoptoticinducing ability of TNF-alpha.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides isolated nucleic acid moleculescomprising, or alternatively consisting of, a nucleic acid sequenceencoding the DR4 polypeptide whose amino acid sequence is shown in SEQID NO:2, or a fragment of the polypeptide. The DR4 polypeptide of thepresent invention shares sequence homology with human TNFR-I, DR3 andFas ligand (FIG. 2). The nucleotide sequence shown in SEQ ID NO:1 wasobtained by sequencing cDNA clones such as HCUDS60, which was depositedon Jan. 21, 1997 at the American Type Culture Collection, 10801University Boulevard, Manassas, Va., 20110-2209, and given AccessionNumber 97853. The deposited clone is contained in the pBK plasmid(Stratagene, La Jolla, Calif.).

Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc.), and allamino acid sequences of polypeptides encoded by DNA molecules determinedherein were predicted by translation of a DNA sequence determined asabove. Therefore, as is known in the art for any DNA sequence determinedby this automated approach, any nucleotide sequence determined hereinmay contain some errors. Nucleotide sequences determined by automationare typically at least about 90% identical, more typically at leastabout 95% to at least about 99.9% identical to the actual nucleotidesequence of the sequenced DNA molecule. The actual sequence can be moreprecisely determined by other approaches including manual DNA sequencingmethods well known in the art. As is also known in the art, a singleinsertion or deletion in a determined nucleotide sequence compared tothe actual sequence will cause a frame shift in translation of thenucleotide sequence such that the predicted amino acid sequence encodedby a determined nucleotide sequence will be completely different fromthe amino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

By “isolated” polypeptide or protein is intended a polypeptide orprotein removed from its native environment. For example, recombinantlyproduced polypeptides and proteins expressed in host cells areconsidered isolated for purposed of the invention as are native orrecombinant polypeptides which have been substantially purified by anysuitable technique such as, for example, the single-step purificationmethod disclosed in Smith and Johnson, Gene 67:31–40 (1988).

Using the information provided herein, such as the nucleic acid sequenceset out in FIGS. 1A–1C, a nucleic acid molecule of the present inventionencoding a DR4 polypeptide may be obtained using standard cloning andscreening procedures, such as those for cloning cDNAs using mRNA asstarting material. Illustrative of the invention, the gene of thepresent invention has also been identified in cDNA libraries of thefollowing tissues: amniotic cells, heart, liver cancer, kidney,leukocyte, activated T-cell, K562 plus PMA, W138 cells, Th2 cells, humantonsils, and CD34 depleted huffy coat (cord blood).

The DR4 gene contains an open reading frame encoding a mature protein ofabout 445 amino acid residues whose initiation codon is at position19–21 of the nucleotide sequence shown in FIGS. 1A–1C (SEQ ID NO:1),with a leader sequence of about 23 amino acid residues (i.e., a totalprotein length of 468 amino acids), and a deduced molecular weight ofabout 50 kDa. Of known members of the TNF receptor family, the DR4polypeptide of the invention shares the greatest degree of homology withhuman TNFR1 and DR3 polypeptides shown in FIG. 2, including significantsequence homology over the multiple Cysteine Rich domains.

In addition to the sequence homology exhibited between DR4 and otherdeath domain containing receptors, DR4 has been shown to bind to TRAILand to induce apoptosis when transiently expressed. MCF7 human breastcarcinoma cells and 293 cells were transiently transfected with a DR4expressing construct, as described in Example 5. As shown in FIGS. 5Aand 5B a substantial proportion of transfected cells underwent themorphological changes characteristic of apoptosis. As anticipated,deletion of the death domain abolished the ability of DR4 to engage thedeath pathway. As can be seen in FIG. 5C, DR4-induced apoptosis wasefficiently blocked by inhibitors of death proteases includingz-VAD-fmk, an irreversible broad spectrum caspase inhibitor and CrmA, acowpox virus encoded serpin that preferentially inhibits apical caspasessuch as FLICE/MACH-1 (caspase-8). Since TNFR-1, CD-95 and DR3-inducedapoptosis is also attenuated by these same inhibitors, it is likely thatthe downstream death effector molecules are similar in nature.

To determine if DR4 was capable of binding TRAIL, the extracellularligand binding domain of DR4 was expressed as a fusion to the Fc regionof human IgG (DR4-Fc). TRAIL selectively bound to DR4-Fc but not tocorresponding extracellular domains of TNFR-1 or CD-95, also expressedas Fc fusions, data not shown. Additionally, DR4-Fc did not bind eitherTNF alpha or Fas ligand under conditions where both of these ligandsbound their cognate receptors.

The ability of TRAIL to induce apoptosis in MCF7 cells was specificallyblocked by DR4-Fc but not influenced by TNFR1-Fc, CD95-Fc or Fc alone(FIG. 6A). Further, as expected, TNF alpha-induced apoptosis wasinhibited by TNFR-1-Fc but not by DR4-Fc, CD95-Fc or Fc alone (FIG. 6B).

Taken together, the data described above indicate that DR4 is a deathdomain containing receptor with the ability to induce apoptosis and is areceptor for TRAIL a known apoptosis inducing ligand.

As indicated, the present invention also provides the mature form(s) ofthe DR4 protein of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal orsecretory leader sequence which is cleaved from the mature protein onceexport of the growing protein chain across the rough endoplasmicreticulum has been initiated. Most mammalian cells and even insect cellscleave secreted proteins with the same specificity. However, in somecases, cleavage of a secreted protein is not entirely uniform, whichresults in two or more mature species on the protein. Further, it haslong been known that the cleavage specificity of a secreted protein isultimately determined by the primary structure of the complete protein,that is, it is inherent in the amino acid sequence of the polypeptide.Therefore, the present invention provides a nucleotide sequence encodingthe mature DR4 polypeptide having the amino acid sequence encoded by thecDNA clones contained in the host identified as ATCC Deposit No. 97853,and as shown in FIGS. 1A–1C (SEQ ID NO:2). By the mature DR4 proteinhaving the amino acid sequence encoded by the cDNA clones contained inthe host identified as ATCC Deposit No. 97853, is meant the matureform(s) of the DR4 protein produced by expression in a mammalian cell(e.g., COS cells, as described below) of the complete open reading frameencoded by the human DNA sequence of the clone contained in the vectorin the deposited host. As indicated below, the mature DR4 having theamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo. 97853, may or may not differ from the predicted “mature” DR4 proteinshown in FIGS. 1A–1C (amino acids from about 24 to about 468) dependingon the accuracy of the predicted cleavage site based on computeranalysis.

Methods for predicting whether a protein has a secretory leader as wellas the cleavage point for that leader sequence are available. Forinstance, the method of McGeoch (Virus Res. 3:271–286 (1985)) and vonHeinje (Nucleic Acids Res. 14:4683–4690 (1986)) can be used. Theaccuracy of predicting the cleavage points of known mammalian secretoryproteins for each of these methods is in the range of 75–80%. vonHeinje, supra. However, the two methods do not always produce the samepredicted cleavage point(s) for a given protein.

In the present case, the predicted amino acid sequence of the completeDR4 polypeptide of the present invention was analyzed by a computerprogram (“PSORT”). (See K. Nakai and M. Kanehisa, Genomics 14:897–911(1992)), which is an expert system for predicting the cellular locationof a protein based on the amino acid sequence. As part of thiscomputational prediction of localization, the methods of McGeoch and vonHeinje are incorporated. The analysis by the PSORT program predicted thecleavage sites between amino acids 23 and 24 in FIG. 1A SEQ ID NO:2.Thereafter, the complete amino acid sequences were further analyzed byvisual inspection, applying a simple form of the (−1,−3) rule of vonHeine. von Heinje, supra. Thus, the leader sequence for the DR4 proteinis predicted to consist of amino acid residues 1–23, underlined in FIG.1A SEQ ID NO:2, while the predicted mature DR4 protein consists of aboutresidues 24–468.

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA may be the coding strand, also known as the sensestrand, or it may be the non-coding strand, also referred to as theanti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its native environmentFor example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the DNA molecules of the presentinvention. Isolated nucleic acid molecules according to the presentinvention further include such molecules produced synthetically.

Isolated nucleic acid molecules of the present invention include DR4 DNAmolecules comprising an open reading frame (ORF) shown in FIGS. 1A–1C(SEQ ID NO:1) and further include DNA molecules which comprise asequence substantially different than all or part of the ORF whoseinitiation codon is at position 19–21 of the nucleotide sequence shownin FIGS. 1A–1C (SEQ ID NO:1) but which, due to the degeneracy of thegenetic code, still encode the DR4 polypeptide or a fragment thereof. Ofcourse, the genetic code is well known in the art. Thus, it would beroutine for one skilled in the art to generate such degenerate variants.

In another aspect, the invention provides isolated nucleic acidmolecules encoding the DR4 polypeptide having an amino acid sequenceencoded by the cDNA clone contained in the plasmid deposited as ATCCDeposit No. 97853 on Jan. 21, 1997. Preferably, these nucleic acidmolecules will encode the mature polypeptide encoded by theabove-described deposited cDNA clone. The invention further provides anisolated nucleic acid molecule having the nucleotide sequence shown inFIGS. 1A–1C (SEQ ID NO:1) or the nucleotide sequence of the DR4 cDNAcontained in the above-described deposited clone, or a nucleic acidmolecule having a sequence complementary to one of the above sequences.Such isolated DNA molecules and fragments thereof are useful as DNAprobes for gene mapping by in situ hybridization of the DR4 gene inhuman tissue by Northern blot analysis.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By fragments of an isolated DNAmolecule having the nucleotide sequence shown in FIGS. 1A–1C (SEQ IDNO: 1) are intended DNA fragments at least 20 bp, and more preferably atleast 30 bp in length which are useful as DNA probes as discussed above,of course larger DNA fragments 50–1500 bp in length are also useful asDNA probes according to the present invention as are DNA fragmentscorresponding to most, if not all, of the nucleotide sequence shown inFIGS. 1A–1C (SEQ ID NO:1). By a fragment at least 20 bp in length, forexample, is intended fragments which include 20 or more bases from thenucleotide sequence in FIGS. 1A–1C (SEQ ID NO:1).

Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding: a polypeptide comprising the DR4extracellular domain (amino acid residues from about 24 to about 238 inFIG. 1A (SEQ ID NO:2)); a polypeptide comprising the DR4 transmembranedomain (amino acid residues from about 239 to about 264 in FIG. 1B (SEQID NO:2)); a polypeptide comprising the DR4 intracellular domain (aminoacid residues from about 265 to about 468 in FIGS. 1B and 1C (SEQ IDNO:2)); and a polypeptide comprising the DR4 death domain (amino acidresidues from about 379 to about 422 in FIG. 1B (SEQ ID NO:2)). Sincethe location of these domains have been predicted by computer graphics,one of ordinary skill would appreciate that the amino acid residuesconstituting these domains may vary slightly (e.g., by about 1 to 15residues) depending on the criteria used to define the domain.

Preferred nucleic acid fragments of the invention encode a full-lengthDR4 polypeptide lacking the nucleotides encoding the amino-terminalmethionine (nucleotides 19–21 in SEQ ID NO:1) as it is known that themethionine is cleaved naturally and such sequences maybe useful ingenetically engineering DR4 expression vectors. Polypeptides encoded bysuch polynucleotides are also contemplated by the invention.

Preferred nucleic acid fragments of the present invention furtherinclude nucleic acid molecules encoding epitope-bearing portions of theDR4 protein. In particular, such nucleic acid fragments of the presentinvention include nucleic acid molecules encoding: a polypeptidecomprising amino acid residues from about 35 to about 92 in FIG. 1A (SEQID NO:2); a polypeptide comprising amino acid residues from about 114 toabout 160 in FIG. 1A (SEQ ID NO:2); a polypeptide comprising amino acidresidues from about 169 to about 240 in FIG. 1A (SEQ ID NO:2); apolypeptide comprising amino acid residues from about 267 to about 298in FIG. 1B (SEQ ID NO:2); a polypeptide comprising amino acid residuesfrom about 330 to about 364 in FIG. 1B (SEQ ID NO:2); a polypeptidecomprising amino acid residues from about 391 to about 404 in FIG. 1B(SEQ ID NO:2); and a polypeptide comprising amino acid residues fromabout 418 to about 465 in FIGS. 1B and 1C (SEQ ID NO:2). The inventorshave determined that the above polypeptide fragments are antigenicregions of the DR4 protein. Methods for determining other suchepitope-bearing portions of the DR4 protein are described in detailbelow.

In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:1 asfollows: HTOIY07R (SEQ ID NO:6) and HTXEY80R (SEQ ID NO:7) both shown inFIG. 4.

Further, the invention includes a polynucleotide comprising any portionof at least about 30 nucleotides, preferably at least about 50nucleotides, of SEQ ID NO:1 from residue 365 to 1,424.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described above, for instance, the cDNAclones contained in ATCC Deposit No. 97853. By “stringent hybridizationconditions” is intended overnight incubation at 42 C in a solutioncomprising: 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in 0.1×SSC at about 65° C.

By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (dither DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30–70 nt of the reference polynucleotide. These are useful asdiagnostic probes and primers as discussed above and in more detailbelow.

By a portion of a polynucleotide of “at least 20 nt in length,” forexample, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNA or the nucleotide sequence as shown in FIGS. 1A–1C (SEQ ID NO:1) orFIGS. 2A–2C (SEQ ID NO:3).

Of course, a polynucleotide which hybridizes only to a poly A sequence(such as the 3 terminal poly(A) tract of the DR4 cDNA shown in FIG. 1C(SEQ ID NO:1)), or to a complementary stretch of T (or U) resides, wouldnot be included in a polynucleotide of the invention used to hybridizeto a portion of a nucleic acid of the invention, since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone).

As indicated, nucleic acid molecules of the present invention whichencode the DR4 polypeptide may include, but are not limited to thecoding sequence for the mature polypeptide, by itself; the codingsequence for the mature polypeptide and additional sequences, such asthose encoding a leader or secretory sequence, such as a pre-, or pro-or prepro-protein sequence; the coding sequence of the maturepolypeptide, with or without the aforementioned additional codingsequences, together with additional, non-coding sequences, including forexample, but not limited to introns and non-coding 5′ and 3′ sequences,such as the transcribed, non-translated sequences that play a role intranscription, mRNA processing including splicing and polyadenylationsignals, for example—ribosome binding and stability of mRNA; additionalcoding sequence which codes for additional amino acids, such as thosewhich provide additional functionalities. Thus, for instance, thepolypeptide may be fused to a marker sequence, such as a peptide, whichfacilitates purification of the fused polypeptide. In certain preferredembodiments of this aspect of the invention, the marker sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(Qiagen, Inc.), among others, many of which are commercially available.As described in Gentz et al., Proc. Natl. Acad Sci. USA 86:821–824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. The HA tag corresponds to an epitopederived of influenza hemagglutinin protein, which has been described byWilson et al., Cell 37:767 (1984), for instance.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode for fragments, analogsor derivatives of the DR4 polypeptide. Variants may occur naturally,such as an allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions which may involve one or more nucleotides. Thevariants may be altered in coding or non-coding regions or both.Alterations in the coding regions may produce conservative ornon-conservative amino acid substitutions, deletions or additions.

Further embodiments of the invention include isolated nucleic acidmolecules that are at least 90% identical, and more preferably at least95%, 96%, 97%, 98% or 99% identical, to (a) a nucleotide sequenceencoding the full-length DR4 polypeptide having the complete amino acidsequence in FIGS. 1A–1C (SEQ ID NO:2), including the predicted leadersequence; (b) nucleotide sequence encoding the full-length DR4polypeptide having the complete amino acid sequence in FIGS. 1A–1C (SEQID NO:2), including the predicted leader sequence but lacking the aminoterminal methionine; (c) a nucleotide sequence encoding the mature DR4polypeptide (full-length polypeptide with the leader removed) having theamino acid sequence at positions about 24 to about 468 in FIGS. 1A–1C(SEQ ID NO:2); (d) a nucleotide sequence encoding the full-length DR4polypeptide having the complete amino acid sequence including the leaderencoded by the cDNA clone contained in ATCC Deposit No. 97853; (e) anucleotide sequence encoding the full-length DR4 polypeptide having thecomplete amino acid sequence including the leader but lacking the aminoterminal methionine encoded by the cDNA clone contained in ATCC DepositNo. 97853; (f) a nucleotide sequence encoding the mature DR4 polypeptidehaving the amino acid sequence encoded by the cDNA clone contained inATCC Deposit No. 97853; (g) a nucleotide sequence that encodes the DR4extracellular domain, (h) a nucleotide sequence that encodes the DR4transmembrane domain, (i) a nucleotide sequence that encodes the DR4intracellular domain, (j) a nucleotide sequence that encodes the DR4death domain; or (k) a nucleotide sequence complementary to any of thenucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), or(j) above.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a DR4polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the DR4polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5 or 3 terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, thenucleotide sequence shown in FIGS. 1A–1C or to the nucleotide sequencesof the deposited cDNA clone can be determined conventionally using knowncomputer programs such as the Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711.Bestfit uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2: 482–489 (1981), to find the bestsegment of homology between two sequences. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

The present application is directed to nucleic acid molecules at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in FIGS. 1A–1C (SEQ ID NO:1) or to the nucleic acid sequence ofthe deposited cDNAs, irrespective of whether they encode a polypeptidehaving DR4 activity. This is because even where a particular nucleicacid molecule does not encode a polypeptide having DR4 activity, one ofskill in the art would still know how to use the nucleic acid molecule,for instance, as a hybridization probe or a polymerase chain reaction(PCR) primer. Uses of the nucleic acid molecules of the presentinvention that do not encode a polypeptide having DR4 activity include,inter alia, (1) isolating the DR4 gene or allelic variants thereof in acDNA library; (2) in situ hybridization (e.g., “FISH”) to metaphasechromosomal spreads to provide precise chromosomal location of the DR4gene, as described in Verma el al., Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York (1988); and (3) Northern Blotanalysis for detecting DR4 mRNA expression in specific tissues.

Preferred, however, are nucleic acid molecules having sequences at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in FIGS. 1A–1C (SEQ ID NO:1) or to the nucleic acid sequence ofthe deposited cDNAs which do, in fact, encode a polypeptide having DR4protein activity. By “a polypeptide having DR4 activity” is intendedpolypeptides exhibiting activity similar, but not necessarily identical,to an activity of the DR4 protein of the invention (either thefull-length protein or, preferably, the mature protein), as measured ina particular biological assay. For example, DR4 protein activity can bemeasured using the cell death assays performed essentially as previouslydescribed (A. M. Chinnaiyan, et al., Cell 81, 505–12 (1995); M. P.Boldin, et al., J Biol Chem 270, 7795–8 (1995); F. C. Kischkel, et al,EMBO 14, 5579–5588 (1995); A. M. Chinnaiyan, et al., J Biol Chem 271,4961–4965 (1996)) or as set forth in Example 5, below. In MCF7 cells,plasmids encoding full-length DR4 or a candidate death domain containingreceptors are co-transfected with the pLantern reporter constructencoding green fluorescent protein. Nuclei of cells transfected with DR4will exhibit apoptotic morphology as assessed by DAPI staining. Similarto TNFR-1 and Fas/APO-1 (M. Muzio, et al., Cell 85, 817–827 (1996); M.P. Boldin, et al., Cell 85, 803–815 (1996); M. Tewari, et al., J BiolChem 270, 3255–60 (1995)), DR4-induced apoptosis is blocked by theinhibitors of ICE-like proteases, CrmA and z-VAD-fmk.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%,98%, or 99% identical to the nucleic acid sequence of the deposited cDNAor the nucleic acid sequence shown in FIGS. 1A–1C (SEQ ID NO:1) willencode a polypeptide “having DR4 protein activity.” In fact, sincedegenerate variants of these nucleotide sequences all encode the samepolypeptide, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving DR4 protein activity. This is because the skilled artisan isfully aware of amino acid substitutions that are either less likely ornot likely to significantly effect protein function (e.g., replacing onealiphatic amino acid with a second aliphatic amino acid).

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U. et al., “Deciphering theMessage in Protein Sequences: Tolerance to Amino Acid Substitutions,”Science 247:1306–1310 (1990), wherein the authors indicate that proteinsare surprisingly tolerant of amino acid substitutions.

Polynucleotide Assays

This invention is also related to the use of the DR4 polynucleotides todetect complementary polynucleotides such as, for example, as adiagnostic reagent. Detection of a mutated form of DR4 associated with adysfunction will provide a diagnostic tool that can add or define adiagnosis of a disease or susceptibility to a disease which results fromunder-expression over-expression or altered expression of DR4 or asoluble form thereof, such as, for example, tumors or autoimmunedisease.

Individuals carrying mutations in the DR4 gene may be detected at theDNA level by a variety of techniques. Nucleic acids for diagnosis may beobtained from a patient's cells, such as from blood, urine, saliva,tissue biopsy and autopsy material. The genomic DNA may be used directlyfor detection or may be amplified enzymatically by using PCR prior toanalysis. (Saiki et al., Nature 324:163–166 (1986)). RNA or cDNA mayalso be used in the same ways. As an example, PCR primers complementaryto the nucleic acid encoding DR4 can be used to identify and analyze DR4expression and mutations. For example, deletions and insertions can bedetected by a change in size of the amplified product in comparison tothe normal genotype. Point mutations can be identified by hybridizingamplified DNA to radiolabeled DR4 RNA or alternatively, radiolabeled DR4antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

Sequence differences between a reference gene and genes having mutationsalso may be revealed by direct DNA sequencing. In addition, cloned DNAsegments may be employed as probes to detect specific DNA segments. Thesensitivity of such methods can be greatly enhanced by appropriate useof PCR or another amplification method. For example, a sequencing primeris used with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels, with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science 230:1242 (1985)).

Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci. USA 85:4397–4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,restriction fragment length polymorphisms (“RFLP”) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations also can be detected by in situ analysis.

Chromosome Assays

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.The mapping of DNAs to chromosomes according to the present invention isan important first step in correlating those sequences with genesassociated with disease.

In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a DR4 gene. This can beaccomplished using a variety of well known techniques and libraries,which generally are available commercially. The genomic DNA the is usedfor in situ chromosome mapping using well known techniques for thispurpose.

In addition, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15–25 bp) from the cDNA. Computer analysis of the 3′untranslated region of the gene is used to rapidly select primers thatdo not span more than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes.

Fluorescence in situ hybridization (“FISH”) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60. For a review of this technique, see Verma et al.,Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, NewYork (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man, available on line through Johns HopkinsUniversity, Welch Medical Library. The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes)).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

Vectors and Host Cells

The present invention also relates to vectors which include DNAmolecules of the present invention, host cells which are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques.

Host cells can be genetically engineered to incorporate nucleic acidmolecules and express polypeptides of the present invention. Thepolynucleotides may be introduced alone or with other polynucleotides.Such other polynucleotides may be introduced independently,co-introduced or introduced joined to the polynucleotides of theinvention.

In accordance with this aspect of the invention the vector may be, forexample, a plasmid vector, a single or double-stranded phage vector, asingle or double-stranded RNA or DNA viral vector. Such vectors may beintroduced into cells as polynucleotides, preferably DNA, by well knowntechniques for introducing DNA and RNA into cells. Viral vectors may bereplication competent or replication defective. In the latter case viralpropagation generally will occur only in complementing host cells.

Preferred among vectors, in certain respects, are those for expressionof polynucleotides and polypeptides of the present invention. Generally,such vectors comprise cis-acting control regions effective forexpression in a host operatively linked to the polynucleotide to beexpressed. Appropriate trans-acting factors either are supplied by thehost, supplied by, a complementing vector or supplied by the vectoritself upon introduction into the host.

A great variety of expression vectors can be used to express apolypeptide of the invention. Such vectors include chromosomal, episomaland virus-derived vectors e.g., vectors derived from bacterial plasmids,from bacteriophage, from yeast episomes, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids, all may be used for expression inaccordance with this aspect of the present invention. Generally, anyvector suitable to maintain, propagate or express polynucleotides toexpress a polypeptide in a host may be used for expression in thisregard.

The DNA sequence in the expression vector is operatively linked toappropriate expression control sequence(s)), including, for instance, apromoter to direct mRNA transcription. Representatives of such promotersinclude the phage lambda PL promoter, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name just a few of the well-known promoters. In general,expression constructs will contain sites for transcription, initiationand termination, and, in the transcribed region, a ribosome binding sitefor translation. The coding portion of the mature transcripts expressedby the constructs will include a translation initiating AUG at thebeginning and a termination codon (UAA, UGA or UAG) appropriatelypositioned at the end of the polypeptide to be translated.

In addition, the constructs may contain control regions that regulate aswell as engender expression. Generally, such regions will operate bycontrolling transcription, such as repressor binding sites andenhancers, among others.

Vectors for propagation and expression generally will include selectablemarkers Such markers also may be suitable for amplification or thevectors may contain additional markers for this purpose. In this regard,the expression vectors preferably contain one or more selectable markergenes to provide a phenotypic trait for selection of transformed hostcells. Preferred markers include dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, and tetracycline or ampicillinresistance genes for culturing E. coli and other bacteria.

The vector containing the appropriate DNA sequence as describedelsewhere herein, as well as an appropriate promoter, and otherappropriate control sequences, may be introduced into an appropriatehost using a variety of well known techniques suitable to expressiontherein of a desired polypeptide. Representative examples of appropriatehosts include bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells. Hosts for of agreat variety of expression constructs are well known, and those ofskill will be enabled by the present disclosure readily to select a hostfor expressing a polypeptides in accordance with this aspect of thepresent invention.

Among vectors preferred for use in bacteria are pQE70, pQE60 and pQE-9,available from Qiagen; pBS vectors, Phagescript vectors, Bluescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; andptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 andpSG available from Stratagene: and pSVK3. pBPV, pMSG and pSVL availablefrom Pharmacia. These vectors are listed solely by way of illustrationof the many commercially available and well known vectors available tothose of skill in the art.

Selection of appropriate vectors and promoters for expression in a hostcell is a well known procedure and the requisite techniques forexpression vector construction, introduction of the vector into the hostand expression in the host are routine skills in the art.

The present invention also relates to host cells containing theabove-described constructs discussed above. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods in MolecularBiology (1986).

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals but also additionalheterologous functional regions. Thus, for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, region also may be added to the polypeptideto facilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilizeproteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of constant regionof immunoglobin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is thoroughlyadvantageous for use in therapy and diagnosis and thus results, forexample, in improved pharmacokinetic properties (EP-A 0232 262). On theother hand, for some uses it would be desirable to be able to delete theFc part after the fusion protein has been expressed, detected andpurified in the advantageous manner described. This is the case when Fcportion proves to be a hindrance to use in therapy and diagnosis, forexample when the fusion protein is to be used as antigen forimmunizations. In drug discovery, for example, human proteins, such as,hIL-5 has been fused with Fc portions for the purpose of high-throughputscreening assays to identify antagonists of hIL-5. See, D. Bennett etal., Journal of Molecular Recognition, Vol. 8:52–58 (1995) and K.Johanson et at., The Journal of Biological Chemistry, Vol. 270, No.16:9459–9471 (1995).

The DR4 polypeptides can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification. Well known techniques forrefolding protein may be employed to regenerate active conformation whenthe polypeptide is denatured during isolation and/or purification.

Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

DR4 polynucleotides and polypeptides may be used in accordance with thepresent invention for a variety of applications, particularly those thatmake use of the chemical and biological properties of DR4. Among theseare applications in treatment of tumors, resistance to parasites,bacteria and viruses, to induce proliferation of T-cells, endothelialcells and certain hematopoietic cells, to treat restenosis, graft vs.host disease, to regulate anti-viral responses and to prevent certainautoimmune diseases after stimulation of DR4 by an agonist. Additionalapplications relate to diagnosis and to treatment of disorders of cells,tissues and organisms. These aspects of the invention are discussedfurther below.

DR4 Polypeptides and Fragments

The invention further provides an isolated DR4 polypeptide having theamino acid sequence shown in FIGS. 1A–1C or a peptide or polypeptidecomprising a portion of the above polypeptides.

To improve or alter the characteristics of DR4 polypeptides, proteinengineering may be employed. Recombinant DNA technology known to thoseskilled in the art can be used to create novel mutant proteins or“muteins including single or multiple amino acid substitutions,deletions, additions or fusion proteins. Such modified polypeptides canshow, e.g., enhanced activity or increased stability. In addition, theymay be purified in higher yields and show better solubility than thecorresponding natural polypeptide, at least under certain purificationand storage conditions.

For instance, for many proteins, including the extracellular domain of amembrane associated protein or the mature form(s) of a secreted protein,it is known in the art that one or more amino acids may be deleted fromthe N-terminus or C-terminus without substantial loss of biologicalfunction. For instance, Ron et al., J. Biol. Chem., 268:2984–2988 (1993)reported modified KGF proteins that had heparin binding activity even if3, 8, or 27 amino-terminal amino acid residues were missing. In thepresent case, since the protein of the invention is a member of thedeath domain containing receptor (DDCR) polypeptide family, deletions ofN-terminal amino acids up to the cysteine residue at position 132 in SEQID NO:2 may retain some biological activity such as the ability toinduce apoptosis. Polypeptides having further N-terminal deletionsincluding the cysteine residue at position 132 (C-132) in SEQ ID NO:2would not be expected to retain such biological activities because thisresidue is conserved among family members, see FIGS. 2A and 2B, and maybe required for forming a disulfide bridge to provide structuralstability which is needed for receptor binding.

However, even if deletion of one or more amino acids from the N-terminusof a protein results in modification of loss of one or more biologicalfunctions of the protein, other biological activities may still beretained. Thus, the ability of the shortened protein to induce and/orbind to antibodies which recognize the complete or extracellular domainof the protein generally will be retained when less than the majority ofthe residues of the complete or extracellular domain protein are removedfrom the N-terminus. Whether a particular polypeptide lacking N-terminalresidues of a complete protein retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the amino acidsequence of DR4 shown in SEQ ID NO:2, up to C-132 residue, andpolynucleotides encoding such polypeptides. In particular, the presentinvention provides polypeptides comprising the amino acid sequence ofresidues n-468 of SEQ ID NO:2, where n is an integer in the range of24–132 where C-132 is the first residue from the N-terminus of theextracellular domain of the DR4 polypeptide (shown in SEQ ID NO:2)believed to be required for receptor-ligand binding (e.g., TRAILbinding) activity of the DR4 protein. Polynucleotides encoding thesepolypeptides are also provided.

Similarly, many examples of biologically functional C-terminal deletionmuteins are known. For instance, interferon gamma shows up to ten timeshigher activities by deleting 8–10 amino acid residues from the carboxyterminus of the protein (Döbeli et al., J. Biotechnology 7:199–216(1988). In the present case, since the protein of the invention is amember of the DDCR polypeptide family, deletions of C-terminal aminoacids up to the cysteine at position 221 (C-221) of SEQ ID NO:2 mayretain some biological activity such receptor binding. Polypeptideshaving further C-terminal deletions including C-221 of SEQ ID NO:2 wouldnot be expected to retain such biological activities because thisresidue is conserved among DDCR family members and is required forforming a disulfide bridge to provide structural stability which isneeded for receptor-ligand binding.

However, even if deletion of one or more amino acids from the C-terminusof a protein results in modification of loss of one or more biologicalfunctions of the protein, other biological activities may still beretained. Thus, the ability of the shortened protein to induce and/orbind to antibodies which recognize the complete or extracellular domainof the protein generally will be retained when less than the majority ofthe residues of the complete or extracellular domain are removed fromthe C-terminus. Whether a particular polypeptide lacking C-terminalresidues of a complete protein retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art.

Accordingly, the present invention further provides polypeptides havingone or more residues from the carboxy terminus of the amino acidsequence of the DR4 shown in SEQ ID NO:2, up to C-221 of SEQ ID NO:2,and polynucleotides encoding such polypeptides. In particular, thepresent invention provides polypeptides having the amino acid sequenceof residues 1-m of the amino acid sequence in SEQ ID NO:2, where m isany integer in the range of 221–468 and residue C-221 is the position ofthe first residue from the C-terminus of the complete DR4 polypeptide(shown in SEQ ID NO:2) believed to be required for receptor bindingactivity of the DR4 protein. Polynucleotides encoding these polypeptidesalso are provided.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini, which may bedescribed generally as having residues n–m of SEQ ID NO:2, where n and mare integers as described above.

Also included are a nucleotide sequence encoding a polypeptideconsisting of a portion of the complete DR4 amino acid sequence encodedby the cDNA clone contained in ATCC Deposit No. 97853, where thisportion excludes from 1 to about 108 amino acids from the amino terminusof the complete amino acid sequence encoded by the cDNA clone containedin ATCC Deposit No. 97853, or from 1 to about 247 amino acids from thecarboxy terminus, or any combination of the above amino terminal andcarboxy terminal deletions, of the complete amino acid sequence encodedby the cDNA clone contained in ATCC Deposit No. 97853. Polynucleotidesencoding all of the above deletion mutant polypeptide forms also areprovided.

Preferred amongst the N- and C-terminal deletion mutants are thosecomprising only a portion of the extracellular domain; i.e., withinresidues 24–238, since any portion therein is expected to be soluble.

It will be recognized in the art that some amino acid sequence of DR4can be varied without significant effect of the structure or function ofthe protein. If such differences in sequence are contemplated, it shouldbe remembered that there will be critical areas on the protein whichdetermine activity. Such areas will usually comprise residues which makeup the ligand binding site or the death domain, or which form tertiarystructures which affect these domains.

Thus, the invention further includes variations of the DR4 protein whichshow substantial DR4 protein activity or which include regions of DR4such as the protein fragments discussed below. Such mutants includedeletions, insertions, inversions, repeats, and type substitutions. Asindicated above, guidance concerning which amino acid changes are likelyto be phenotypically silent can be found in Bowie, J. U. et al., Science247:1306–1310 (1990).

Of particular interest are substitutions of charged amino acids withanother charged amino acid and with neutral or negatively charged aminoacids. The latter results in proteins with reduced positive charge toimprove the characteristics of the DR4 protein. The prevention ofaggregation is highly desirable. Aggregation of proteins not onlyresults in a loss of activity but can also be problematic when preparingpharmaceutical formulations, because they can be immunogenic. (Pinckardet al., Clin Exp. Immunol. 2:331–340 (1967); Robbins et al., Diabetes36:838–845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug CarrierSystems 10:307–377 (1993)).

The replacement of amino acids can also change the selectivity ofbinding to cell surface receptors. Ostade et al., Nature 361:266–268(1993) describes certain mutations resulting in selective binding ofTNF-alpha to only one of the two known types of TNF receptors. Thus, theDR4 receptor of the present invention may include one or more amino acidsubstitutions, deletions or additions, either from natural mutations orhuman manipulation.

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table 1).

TABLE 1 Conservative Amino Acid Substitutions. Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

Amino acids in the DR4 protein of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081–1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding or in vitro, or in vitro proliferativeactivity. Sites that are critical for ligand-receptor binding can alsobe determined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al., J. Mol.Biol. 224:899–904 (1992) and de Vos et al. Science 255:306–312 (1992)).

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of the DR4 polypeptide is substantiallypurified by the one-step method described in Smith and Johnson, Gene67:31–40 (1988).

The polypeptides of the present invention also include the polypeptideencoded by the deposited cDNA including the leader, the maturepolypeptide encoded by the deposited the cDNA minus the leader (i.e.,the mature protein), the polypeptide of FIGS. 1A–1C (SEQ ID NO:2)including the leader, the polypeptide of FIGS. 1A–1C (SEQ ID NO:2) minusthe amino terminal methionine, the polypeptide of FIGS. 1A–1C (SEQ ID NO:2) minus the leader, the extracellular domain, the transmembranedomain, the intracellular domain, the death domain, soluble polypeptidescomprising all or part of the extracellular and intracelluar domains butlacking the transmembrane domain as well as polypeptides which are atleast 80% identical, more preferably at least 90% or 95% identical,still more preferably at least 96%, 97%, 98% or 99% identical to thepolypeptide encoded by the deposited cDNA clones, to the polypeptide ofFIGS. 1A–1C (SEQ ID NO:2) and also include portions of such polypeptideswith at least 30 amino acids and more preferably at least 50 aminoacids.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a DR4 polypeptideis intended that the amino acid sequence of the polypeptide is identicalto the reference sequence except that the polypeptide sequence mayinclude up to five amino acid alterations per each 100 amino acids ofthe reference amino acid of the DR4 polypeptide. In other words, toobtain a polypeptide having an amino acid sequence at least 95%identical to a reference amino acid sequence, up to 5% of the amino acidresidues in the reference sequence may be deleted or substituted withanother amino acid, or a number of amino acids up to 5% of the totalamino acid residues in the reference sequence may be inserted into thereference sequence. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequence shown in FIGS. 1A–1C (SEQ ID NO:2) or to the amino acidsequence encoded by deposited cDNA clones can be determinedconventionally using known computer programs such the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711. When using Bestfit or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

The present inventors have discovered that the DR4 polypeptide is a 468residue protein exhibiting three main structural domains. First, theligand binding domain was identified within residues from about 24 toabout 238 in FIGS. 1A and 1B. Second, the transmembrane domain wasidentified within residues from about 239 to about 264 in FIG. 1B.Third, the intracellular domain was identified within residues fromabout 265 to about 468 in FIGS. 1B and 1C. Importantly, theintracellular domain includes a death domain at residues from about 379to about 422. Further preferred fragments of the polypeptide shown inFIGS. 1A–1C include the mature protein from residues about 24 to about468 and soluble polypeptides comprising all or part of the extracellularand intracellular domains but lacking the transmembrane domain.

The invention further provides DR4 polypeptides encoded by the depositedcDNA clone including the leader and DR4 polypeptide fragments selectedfrom the mature protein, the extracellular domain, the transmembranedomain, the intracellular domain, and the death domain.

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide described herein.The epitope of this polypeptide portion is an immunogenic or antigenicepitope of a polypeptide of the invention. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response whenthe whole protein is the immunogen. On the other hand, a region of aprotein molecule to which an antibody can bind is defined as an“antigenic epitope.” The number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes. See, forinstance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998–4002 (1983).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M.,Green, N. and Learner, R. A. (1983) Antibodies that react withpredetermined sites on proteins. Science 219:660–666. Peptides capableof eliciting protein-reactive sera are frequently represented in theprimary sequence of a protein, can be characterized by a set of simplechemical rules, and are confined neither to immunodominant regions ofintact proteins (i.e., immunogenic epitopes) nor to the amino orcarboxyl terminals.

Antigenic epitope-bearing peptides and polypeptides of the invention aretherefore useful to raise antibodies, including monoclonal antibodies,that bind specifically to a polypeptide of the invention. See, forinstance, Wilson et al., Cell 37:767–778 (1984) at 777.

Antigenic epitope-bearing peptides and polypeptides of the inventionpreferably contain a sequence of at least seven, more preferably atleast nine and most preferably between at least about 15 to about 30amino acids contained within the amino acid sequence of a polypeptide ofthe invention.

Non-limiting examples of antigenic polypeptides or peptides that can beused to generate DR4-specific antibodies include: a polypeptidecomprising amino acid residues from about 35 to about 92 in FIG. 1A (SEQID NO:2); a polypeptide comprising amino acid residues from about 114 toabout 160 in FIG. 1A (SEQ ID NO:2); a polypeptide comprising amino acidresidues from about 169 to about 240 in FIGS. 1A and 1B (SEQ ID NO:2); apolypeptide comprising amino acid residues from about 267 to about 298in FIG. 1B (SEQ ID NO:2); a polypeptide comprising amino acid residuesfrom about 330 to about 364 in FIG. 1B (SEQ ID NO:2); a polypeptidecomprising amino acid residues from about 391 to about 404 in FIGS. 1Band 1C (SEQ ID NO:2); and a polypeptide comprising amino acid residuesfrom about 418 to about 465 in FIG. 1C (SEQ ID NO:2). As indicatedabove, the inventors have determined that the above polypeptidefragments are antigenic regions of the DR4 protein.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means. Houghten, R. A., “General method forthe rapid solid-phase synthesis of large numbers of peptides:specificity of antigen-antibody interaction at the level of individualamino acids,” Proc. Natl. Acad. Sci. USA 82:5131–5135 (1985). This“Simultaneous Multiple Peptide Synthesis (SMPS)” process is furtherdescribed in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).

As one of skill in the art will appreciate, DR4 polypeptides of thepresent invention and the epitope-bearing fragments thereof describedabove can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been shown, e.g., for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84–86(1988)). Fusion proteins that have a disulfide-linked dimeric structuredue to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric DR4 protein or proteinfragment alone (Fountoulakis et al., J Biochem 270:3958–3964 (1995)).

Polypeptide Assays

The present invention also relates to diagnostic assays such asquantitative and diagnostic assays for detecting levels of DR4 protein,or the soluble form thereof, in cells and tissues, includingdetermination of normal and abnormal levels. Thus, for instance, adiagnostic assay in accordance with the invention for detectingover-expression of DR4, or soluble form thereof, compared to normalcontrol tissue samples may be used to detect the presence of tumors, forexample. Assay techniques that can be used to determine levels of aprotein, such as a DR4 protein of the present invention, or a solubleform thereof, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

Assaying DR4 protein levels in a biological sample can occur using anyart-known method. Preferred for assaying DR4 protein levels in abiological sample are antibody-based techniques. For example, DR4protein expression in tissues can be studied with classicalimmunohistological methods. (Jalkanen, M., et al., J. Cell. Biol.101:976–985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087–3096(1987)).

Other antibody-based methods useful for detecting DR4 protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA).

Suitable labels are known in the art and include enzyme labels, such asglucose oxidase, radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon(¹⁴C), sulphur (³⁵S), tritium (³H), indium (¹¹²In), and technetium(^(99m)Tc), and fluorescent labels, such as fluorescein and rhodamine,and biotin.

Therapeutics

The Tumor Necrosis Factor (TNF) family ligands are known to be among themost pleiotropic cytokines, inducing a large number of cellularresponses, including cytotoxicity, anti-viral activity, immunoregulatoryactivities, and the transcriptional regulation of several genes(Goeddel, D. V. et al., “Tumor Necrosis Factors: Gene Structure andBiological Activities,” Symp. Quani. Biol. 51:597–609 (1986), ColdSpring Harbor; Beutler, B., and Cerami, A., Annu. Rev. Biochem.57:505–518 (1988); Old, L. J., Sci. Am. 258:59–75 (1988); Fiers, W.,FEBS Lett. 285:199–224 (1991)). The TNF-family ligands induce suchvarious cellular responses by binding to TNF-family receptors, includingthe DR4 receptors of the present invention. Cells which express the DR4polypeptide and are believed to have a potent cellular response to DR4ligand include amniotic cells, heart, liver cancer kidney, peripheralblood leukocytes, activated T-cells, tissue corresponding to Th2 cells,human tonsils, and CD34 depleted buffy coat (cord blood). By “a cellularresponse to a TNF-family ligand” is intended any genotypic, phenotypic,and/or morphologic change to a cell, cell line, tissue, tissue cultureor patient that is induced by a TNF-family ligand. As indicated, suchcellular responses include not only normal physiological responses toTNF-family ligand, but also diseases associated with increased apoptosisor the inhibition of apoptosis. Apoptosis-programmed cell death-is aphysiological mechanism involved in the deletion of peripheral Tlymphocytes of the immune system, and its dysregulation can lead to anumber of different pathogenic processes (Ameisen, J. C., AXDS8:1197–1213 (1994); Krammer, P. H. et al., Curr. Opin. Immunol.6:279–289 (1994)).

Diseases associated with increased cell survival, or the inhibition ofapoptosis, include cancers (such as follicular lymphomas, carcinomaswith p53 mutations, and hormone-dependent tumors, such as breast cancer,prostrate cancer, Kaposi's sarcoma and ovarian cancer); autoimmunedisorders (such as systemic lupus erythematosus and immune-relatedglomerulonephritis rheumatoid arthritis) and viral infections (such asherpes viruses, pox viruses and adenoviruses), information graft v. hostdisease, acute graft rejection, and chronic graft rejection. Diseasesassociated with increased apoptosis include AIDS; neurodegenerativedisorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophiclateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration);myelodysplastic syndromes (such as aplastic anemia), ischemic injury(such as that caused by myocardial infarction, stroke and reperfusioninjury), toxin-induced liver disease (such as that caused by alcohol),septic shock, cachexia and anorexia.

Thus, in one aspect, the present invention is directed to a method forenhancing apoptosis induced by a TNF-family ligand, which involvesadministering to a cell which expresses the DR4 polypeptide an effectiveamount of DR4 ligand, analog or an agonist capable of increasing DR4mediated signaling. Preferably, DR4 mediated signaling is increased totreat a disease wherein decreased apoptosis or decreased cytokine andadhesion molecule expression is exhibited. An agonist can includesoluble forms of DR4 and monoclonal antibodies directed against the DR4polypeptide.

In a further aspect, the present invention is directed to a method forinhibiting apoptosis induced by a TNF-family ligand, which involvesadministering to a cell which expresses the DR4 polypeptide an effectiveamount of an antagonist capable of decreasing DR4 mediated signaling.Preferably, DR4 mediated signaling is decreased to treat a diseasewherein increased apoptosis or NFkB expression is exhibited. Anantagonist can include soluble forms of DR4 and monoclonal antibodiesdirected against the DR4 polypeptide. exhibited. An antagonist caninclude soluble forms of DR4 and monoclonal antibodies directed againstthe DR4 polypeptide.

By “agonist” is intended naturally occurring and synthetic compoundscapable of enhancing or potentiating apoptosis. By “antagonist” isintended naturally occurring and synthetic compounds capable ofinhibiting apoptosis. Whether any candidate “agonist” or “antagonist” ofthe present invention can enhance or inhibit apoptosis can be determinedusing art-known TNF-family ligand/receptor cellular response assays,including those described in more detail below.

One such screening procedure involves the use of melanophores which aretransfected to express the receptor of the present invention. Such ascreening technique is described in PCT WO 92/01810, published Feb. 6,1992. Such an assay may be employed, for example, for screening for acompound which inhibits (or enhances) activation of the receptorpolypeptide of the present invention by contacting the melanophore cellswhich encode the receptor with both a TNF-family ligand and thecandidate antagonist (or agonist). Inhibition or enhancement of thesignal generated by the ligand indicates that the compound is anantagonist or agonist of the ligand/receptor signaling pathway.

Other screening techniques include the use of cells which express thereceptor (for example, transfected CHO cells) in a system which measuresextracellular pH changes caused by receptor activation, for example, asdescribed in Science 246:181–296 (October 1989). For example, compoundsmay be contacted with a cell which expresses the receptor polypeptide ofthe present invention and a second messenger response, e.g., signaltransduction or pH changes, may be measured to determine whether thepotential compound activates or inhibits the receptor.

Another such screening technique involves introducing RNA encoding thereceptor into Xenopus oocytes to transiently express the receptor. Thereceptor oocytes may then be contacted with the receptor ligand and acompound to be screened, followed by detection of inhibition oractivation of a calcium signal in the case of screening for compoundswhich are thought to inhibit activation of the receptor.

Another screening technique involves expressing in cells a constructwherein the receptor is linked to a phospholipase C or D. Such cellsinclude endothelial cells, smooth muscle cells, embryonic kidney cells,etc. The screening may be accomplished as hereinabove described bydetecting activation of the receptor or inhibition of activation of thereceptor from the phospholipase signal.

Another method involves screening for compounds which inhibit activationof the receptor polypeptide of the present invention antagonists bydetermining inhibition of binding of labeled ligand to cells which havethe receptor on the surface thereof. Such a method involves transfectinga eukaryotic cell with DNA encoding the receptor such that the cellexpresses the receptor on its surface and contacting the cell with acompound in the presence of a labeled form of a known ligand. The ligandcan be labeled, e.g., by radioactivity. The amount of labeled ligandbound to the receptors is measured, e.g., by measuring radioactivity ofthe receptors. If the compound binds to the receptor as determined by areduction of labeled ligand which binds to the receptors, the binding oflabeled ligand to the receptor is inhibited.

Further screening assays for agonist and antagonist of the presentinvention are described in Tartaglia, L. A., and Goeddel, D. V., J.Biol. Chem. 267(7):4304–4307 (1992).

Thus, in a further aspect, a screening method is provided fordetermining whether a candidate agonist or antagonist is capable ofenhancing or inhibiting a cellular response to a TNF-family ligand. Themethod involves contacting cells which express the DR4 polypeptide witha candidate compound and a TNF-family ligand, assaying a cellularresponse, and comparing the cellular response to a standard cellularresponse, the standard being assayed when contact is made with theligand in absence of the candidate compound, whereby an increasedcellular response over the standard indicates that the candidatecompound is an agonist of the ligand/receptor signaling pathway and adecreased cellular response compared to the standard indicates that thecandidate compound is an antagonist of the ligand/receptor signalingpathway. By “assaying a cellular response” is intended qualitatively orquantitatively measuring a cellular response to a candidate compoundand/or a TNF-family ligand (e.g., determining or estimating an increaseor decrease in T cell proliferation or tritiated thymidine labeling). Bythe invention, a cell expressing the DR4 polypeptide can be contactedwith either an endogenous or exogenously administered TNF-family ligand.

Agonist according to the present invention include naturally occurringand synthetic compounds such as, for example, TNF family ligand peptidefragments, transforming growth factor, neurotransmitters (such asglutamate, dopamine, N-methyl-D-aspartate), tumor suppressors (p53),cytolytic T cells and antimetabolites. Preferred agonist includechemotherapeutic drugs such as, for example, cisplatin, doxorubicin,bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate andvincristine. Others include ethanol and -amyloid peptide. (Science267:1457–1458 (1995)). Further preferred agonist include polyclonal andmonoclonal antibodies raised against the DR4 polypeptide, or a fragmentthereof. Such agonist antibodies raised against a TNF-family receptorare disclosed in Tartaglia, L. A., et al., Proc. Natl. Acad. Sci. USA88:9292–9296 (1991); and Tartaglia, L. A., and Goeddel, D. V., J. Biol.Chem. 267 (7):4304–4307 (1992) See, also, PCT Application WO 94/09137.

Antagonist according to the present invention include naturallyoccurring and synthetic compounds such as, for example, the CD40 ligand,neutral amino acids, zinc, estrogen, androgens, viral genes (such asAdenovirus ElB, Baculovirus p35 and IAP, Cowpox virus crmA, Epstein-Barrvirus BHRF1, LMP-1, African swine fever virus LMW5-HL, and HerpesvirusICP 34.5), calpain inhibitors, cysteine protease inhibitors, and tumorpromoters (such as PMA, Phenobarbital, and hexachlorocyclohexanes (e.g.,α-, β-, or γ-hexachlorocyclohexane).

Other potential antagonists include antisense molecules. Antisensetechnology can be used to control gene expression through antisense DNAor RNA or through triple-helix formation. Antisense techniques arediscussed, for example, in Okano, J. Neurochem. 56:560 (1991);Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988). Triple helix formation is discussed in,for instance Lee et al., Nucleic Acids Research 6:3073 (1979); Cooney etal., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991).The methods are based on binding of a polynucleotide to a complementaryDNA or RNA.

For example, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide. The oligonucleotidesdescribed above can also be delivered to cells such that the antisenseRNA or DNA may be expressed in vivo to inhibit production of thereceptor.

Further antagonist according to the present invention include solubleforms of DR4, i.e., DR4 fragments that include the ligand binding domainfrom the extracellular region of the full length receptor. Such solubleforms of the receptor, which may be naturally occurring or synthetic,antagonize DR4 mediated signaling by competing with the cell surface DR4for binding to TNF-family ligands. Thus, soluble forms of the receptorthat include the ligand binding domain are novel cytokines capable ofinhibiting apoptosis induced by TNF-family ligands. These are preferablyexpressed as dimers or trimers, since these have been shown to besuperior to monomeric forms of soluble receptor as antagonists, e.g.,IgGFc-TNF receptor family fusions. Other such cytokines are known in theart and include Fas B (a soluble form of the mouse Fas receptor) thatacts physiologically to limit apoptosis induced by Fas ligand (Hughes,D. P. and Crispe, I. N., J. Exp. Med. 182:1395–1401 (1995)).

The experiments set forth in Example 5 demonstrates that DR4 is a deathdomain-containing molecule capable of triggering apoptosis which isimportant in the regulation of the immune system. In addition, theexperiments set forth below demonstrate that DR4-induced apoptosis wasblocked by the inhibitors of ICE-like proteases, CrmA and z-VAD-fmk.Thus, inhibitors of ICE-like proteases, FADD-DN and FLICE-DN/MACHa1C360Scould also be used as antagonists for DR4 activity.

The term “antibody” (Ab) or “monoclonal antibody” (mAb) as used hereinis meant to include intact molecules as well as fragments thereof (suchas, for example, Fab and F(ab′)₂ fragments) which are capable of bindingan antigen. Fab and F(ab′)₂ fragments lack the Fc fragment of intactantibody, clear more rapidly from the circulation, and may have lessnon-specific tissue binding of an intact antibody (Wahl et al., J. Nucl.Med. 24:316–325 (1983)).

Antibodies according to the present invention may be prepared by any ofa variety of methods using DR4 immunogens of the present invention. Asindicated, such DR4 immunogens include the full length DR4 polypeptide(which may or may not include the leader sequence) and DR4 polypeptidefragments such as the ligand binding domain, the transmembrane domain,the intracellular domain and the death domain.

Proteins and other compounds which bind the DR4 domains are alsocandidate agonist and antagonist according to the present invention.Such binding compounds can be “captured” using the yeast two-hybridsystem (Fields and Song, Nature 340:245–246 (1989)). A modified versionof the yeast two-hybrid system has been described by Roger Brent and hiscolleagues (Gyuris, J. et al, Cell 75:791–803 (1993); Zervos, A. S. etal., Cell 72:223–232 (1993)). Preferably, the yeast two-hybrid system isused according to the present invention to capture compounds which bindto either the DR4 ligand binding domain or to the DR4 intracellulardomain. Such compounds are good candidate agonist and antagonist of thepresent invention.

By a “TNF-family ligand” is intended naturally occurring, recombinant,and synthetic ligands that are capable of binding to a member of the TNFreceptor family and inducing and/or blocking the ligand/receptorsignaling pathway. Members of the TNF ligand family include, but are notlimited to, DR4 ligands, TRAIL, TNF-α, TNF-β-α, lymphotoxin-α (LT-α,also known as TNF-β), LT-β (found in complex heterotrimer LT-α2-β),FasL, VEGI (International Publication No. WO 96/14328), AIM-I(International Publication No. WO 97/33 899), AIM-II (InternationalPublication No. WO 97/34911), APRIL (J. Exp. Med. 188(6):1185–1190),endokine-alpha (International Publication No. WO 98/07880),neutrokine-alpha (International Publication No. WO 98/18921), CD40L,CD27L, CD30L, 4-1BBL, OX40L and nerve growth factor (NGF).

Representative therapeutic applications of the present invention arediscussed in-more detail below. The state of immunodeficiency thatdefines AIDS is secondary to a decrease in the number and function ofCD4⁺ T-lymphocytes. Recent reports estimate the daily loss of CD4⁺ Tcells to be between 3.5×10⁷ and 2×10⁹ cells (Wei X., et al., Nature373:117–122 (1995)). One cause of CD4⁺ T cell depletion in the settingof HIV infection is believed to be HIV-induced apoptosis. Indeed,HIV-induced apoptotic cell death has been demonstrated not only in vitrobut also, more importantly, in infected individuals (Ameisen, J. C.,AIDS 8:1197–1213 (1994); Finkel, T. H., and Banda, N. K., Curr. Opin.Immunol. 6:605–615 (1995); Muro-Cacho, C. A. et al., J. Immunol.154:5555–5566 (1995)). Furthermore, apoptosis and CD4⁺ T-lymphocytedepletion is tightly correlated in different animal models of AIDS(Brunner, T., et al., Nature 373:441–444 (1995); Gougeon, M. L., et al.,AIDS Res. Hum. Retroviruses 9:553–563 (1993)) and, apoptosis is notobserved in those animal models in which viral replication does notresult in AIDS (Gougeon, M. L. et al., AIDS Res. Hum. Retroviruses9:553–563 (1993)). Further data indicates that uninfected but primed oractivated T lymphocytes from HIV-infected individuals undergo apoptosisafter encountering the TNF-family ligand FasL. Using monocytic celllines that result in death following HIV infection, it has beendemonstrated that infection of U937 cells with HIV results in the denovo expression of FasL and that FasL mediates HIV-induced apoptosis(Badley, A. D. et al., J. Virol. 70:199–206 (1996)). Further theTNF-family ligand was detectable in uninfected macrophages and itsexpression was upregulated following HIV infection resulting inselective killing of uninfected CD4 T-lymphocytes (Badley, A. D et al.,J. Virol. 70:199–206 (1996)). Thus, by the invention, a method fortreating HIV⁺ individuals is provided which involves administering anantagonist of the present invention to reduce selective killing of CD4T-lymphocytes. Modes of administration and dosages are discussed indetail below.

In rejection of an allograft, the immune system of the recipient animalhas not previously been primed to respond because the immune system forthe most part is only primed by environmental antigens. Tissues fromother members of the same species have not been presented in the sameway that, for example, viruses and bacteria have been presented. In thecase of allograft rejection, immunosuppressive regimens are designed toprevent the immune system from reaching the effector stage. However, theimmune profile of xenograft rejection may resemble disease recurrencemore that allograft rejection. In the case of disease recurrence, theimmune system has already been activated, as evidenced by destruction ofthe native islet cells. Therefore, in disease recurrence the immunesystem is already at the effector stage. Agonist of the presentinvention are able to suppress the immune response to both allograftsand xenografts because lymphocytes activated and differentiated intoeffector cells will express the DR4 polypeptide, and thereby aresusceptible to compounds which enhance apoptosis. Thus, the presentinvention further provides a method for creating immune privilegedtissues. Antagonist of the invention can further be used in thetreatment of Inflammatory Bowel-Disease.

DR4 antagonists may be useful for treating inflammatory diseases, suchas rheumatoid arthritis, osteoarthritis, psoriasis, septicemia, andinflammatory bowel disease.

In addition, due to lymphoblast expression of DR4, soluble DR4, agonistor antagonist mABs may be used to treat this form of cancer. Further,soluble DR4 or neutralizing mABs may be used to treat various chronicand acute forms of inflammation such as rheumatoid arthritis,osteoarthritis, psoriasis, septicemia, and inflammatory bowel disease.

Modes of Administration

The agonist or antagonists described herein can be administered invitro, ex vivo, or in vivo to cells which express the receptor of thepresent invention. By administration of an “effective amount” of anagonist or antagonist is intended an amount of the compound that issufficient to enhance or inhibit a cellular response to a TNF-familyligand and include polypeptides. In particular, by administration of an“effective amount” of an agonist or antagonists is intended an amounteffective to enhance or inhibit DR4 mediated apoptosis. Of course, whereapoptosis is to be enhanced, an agonist according to the presentinvention can be co-administered with a TNF-family ligand. One ofordinary skill will appreciate that effective amounts of an agonist orantagonist can be determined empirically and may be employed in pureform or in pharmaceutically acceptable salt, ester or prodrug form. Theagonist or antagonist may be administered in compositions in combinationwith one or more pharmaceutically acceptable excipients.

It will be understood that, when administered to a human patient, thetotal daily usage of the compounds and compositions of the presentinvention will be decided by the attending physician within the scope ofsound medical judgement. The specific therapeutically effective doselevel for any particular patient will depend upon factors well known inthe medical arts.

As a general proposition, the total pharmaceutically effective amount ofDR4 polypeptide administered parenterally per dose will be in the rangeof about 1 μg/kg/day to 10 mg/kg/day of patient body weight, although,as noted above, this will be subject to therapeutic discretion. Morepreferably, this dose is at least 0.01 mg/kg/day, and most preferablyfor humans between about 0.01 and 1 mg/kg/day for the hormone. If givencontinuously, the DR4 agonists or antagonists is typically administeredat a dose rate of about 1 μg/kg/hour to about 50 μg/kg/hour, either by1–4 injections per day or by continuous subcutaneous infusions, forexample, using a mini-pump. An intravenous bag solution may also beemployed.

Dosaging may also be arranged in a patient specific manner to provide apredetermined concentration of an agonist or antagonist in the blood, asdetermined by the RIA technique. Thus patient dosaging may be adjustedto achieve regular on-going trough blood levels, as measured by RIA, onthe order of from 50 to 1000 ng/ml, preferably 150 to 500 ng/ml.

Pharmaceutical compositions are provided comprising an agonist orantagonist and a pharmaceutically acceptable carrier or excipient, whichmay be administered orally, rectally, parenterally, intracistemally,intravaginally, intraperitoneally, topically (as by powders, ointments,drops or transdermal patch), bucally, or as an oral or nasal spray.Importantly, by co-administering an agonist and a TNF-family ligand,clinical side effects can be reduced by using lower doses of both theligand and the agonist. It will be understood that the agonist can be“co-administered” either before, after, or simultaneously with theTNF-family ligand, depending on the exigencies of a particulartherapeutic application. By “pharmaceutically acceptable carrier” ismeant a non-toxic solid, semisolid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. The term“parenteral” as used herein refers to modes of administration whichinclude intravenous, intramuscular, intraperitoneal, intrastemal,subcutaneous and intraarticular injection and infusion.

Pharmaceutical compositions of the present invention for parenteralinjection can comprise pharmaceutically acceptable sterile aqueous ornonaqueous solutions, dispersions, suspensions or emulsions as well assterile powders for reconstitution into sterile injectable solutions ordispersions just prior to use.

In addition to soluble DR4 polypeptides, DR4 polypeptide containing thetransmembrane region can also be used when appropriately solubilized byincluding detergents, such as CHAPS or NP40, with buffer.

EXAMPLE 1 Expression and Purification in E. coli

The DNA sequence encoding the mature DR4 protein in the deposited cDNAclone (ATCC No. 97853) is amplified using PCR oligonucleotide primersspecific to the amino terminal sequences of the DR4 protein and tovector sequences 3′ to the gene. Additional nucleotides containingrestriction sites to facilitate cloning are added to the 5′ and 3′sequences respectively.

The following primers are used for expression of DR4 extracellulardomain in E. coli 5′ primer 5′-GCGGCATGCATGATCAATCAATTGGCAC-3′ (SEQ IDNO:8) contains the underlined SphI site. 3′ primer:

5′-GCGAAGCTTTCAATTATGTCCATTGCCTG-3′ (SEQ ID NO:12) contains theunderlined HindIII site. Vector is pQE60.

The restriction sites are convenient to restriction enzyme sites in thebacterial expression vector pQE60, which are used for bacterialexpression in these examples. (Qiagen, Inc. 9259 Eton Avenue,Chatsworth, Calif., 91311). pQE60 encodes ampicillin antibioticresistance (“Amp”) and contains a bacterial origin of replication(“ori”), an IPTG inducible promoter, a ribosome binding site (“RBS”).

The amplified DR4 DNA and the vector pQE60 both are digested with SphIand HindIII and the digested DNAs are then ligated together. Insertionof the DDCR protein DNA into the restricted pQE60 vector places the DR4protein coding region downstream of and operably linked to the vector'sIPTG-inducible promoter and in-frame with an initiating AUGappropriately positioned for translation of DR4 protein.

The ligation mixture is transformed into competent E. coli cells usingstandard procedures. Such procedures are described in Sambrook et al.,Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses lac repressor and confers kanamycin resistance (“Kan^(r)”), isused in carrying out the illustrative example described herein. Thisstrain, which is only one of many that are suitable for expressing DR4protein, is available commercially from Qiagen.

Transformants are identified by their ability to grow on LB plates inthe presence of ampicillin and kanamycin. Plasmid DNA is isolated fromresistant colonies and the identity of the cloned DNA confirmed byrestriction analysis.

Clones containing the desired constructs are grown overnight (“ON”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml).

The O/N culture is used to inoculate a large culture, at a dilution ofapproximately 1:100 to 1:250. The cells are grown to an optical densityat 600 nm (“OD600”) of between 0.4 and 0.6.Isopropyl-B-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from lac repressorsensitive promoters, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation and disrupted, by standard methods.Inclusion bodies are purified from the disrupted cells using routinecollection techniques, and protein is solubilized from the inclusionbodies into 8M urea. The 8M urea solution containing the solubilizedprotein is passed over a PD-10 column in 2 × phosphate-buffered saline(“PBS”), thereby removing the urea, exchanging the buffer and refoldingthe protein. The protein is purified by a further step of chromatographyto remove endotoxin. Then, it is sterile filtered. The sterile filteredprotein preparation is stored in 2×PBS at a concentration of 95 μ/ml.

EXAMPLE 2 Expression in Mammalian Cells

Most of the vectors used for the transient expression of a given genesequence in mammalian cells carry the SV40 origin of replication. Thisallows the replication of the vector to high copy numbers in cells (e.g.COS cells) which express the T antigen required for the initiation ofviral DNA synthesis. Any other mammalian cell line can also be utilizedfor this purpose.

A typical mammalian expression vector contains the promoter element,which mediates the initiation of transcription of mRNA, the proteincoding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRs) from Retroviruses, e.g. RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, also cellular signals can be used (e.g., human actin,promoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC67109). Mammalian host cells that could be usedinclude, human HeLa, 283, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV1 African green monkey cells, quail QC1–3cells, mouse L cells and Chinese hamster ovary cells.

Alternatively, a gene of interest can be expressed in stable cell linesthat contain the gene integrated into a chromosome. The co-transfectionwith a selectable marker such as dhfr, gpt, neomycin, hygromycin allowsthe identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) is a usefulmarker to develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Using this marker, themammalian cells are grown in increasing amounts of methotrexate forselection and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) cells are often used for the production ofproteins.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology438:44701 (March 1985)), plus a fragment of the CMV-enhancer (Boshart etal., Cell 41:521–530 (1985)). Multiple cloning sites, e.g. with therestriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate thecloning of the gene of interest. The vectors contain in addition the 3′intron, the polyadenylation and termination signal of the ratpreproinsulin gene.

Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of DR4 polypeptide. PlasmidpC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146).The plasmid contains the mouse DHFR gene under control of the SV40 earlypromoter. Chinese hamster ovary- or other cells lacking dihydrofolateactivity that are transfected with these plasmids can be selected bygrowing the cells in a selective medium (alpha minus MEM, LifeTechnologies) supplemented with the chemotherapeutic agent methotrexate.The amplification of the DHFR genes in cells resistant to methotrexate(MTX) has been well documented (see, e.g., Alt, F. W., Kellems, R. M.,Bertino, J. R., and Schimke, R. T., 1978, J. Biol. Chem. 253:1357–1370,Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta, 1097:107–143,Page, M. J. and Sydenham, M. A. 1991, Biotechnology 9:64–68). Cellsgrown in increasing concentrations of MTX develop resistance to the drugby overproducing the target enzyme, DHFR, as a result of amplificationof the DHFR gene. If a second gene is linked to the DHFR gene, it isusually co-amplified and over-expressed. It is known in the art thatthis approach may be used to develop cell lines carrying more than 1,000copies of the amplified gene(s). Subsequently, when the methotrexate iswithdrawn, cell lines are obtained which contain the amplified geneintegrated into one or more chromosome(s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strongpromoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus(Cullen, et al., Molecular and Cellular Biology, March 1985:438–447)plus a fragment isolated from the enhancer of the immediate early geneof human cytomegalovirus (CMV) (Boshart et al., Cell 41:521–530 (1985)).Downstream of the promoter are the following single restriction enzymecleavage sites that allow the integration of the genes: BamHI, Xba I,and Asp718. Behind these cloning sites the plasmid contains the 3′intron and polyadenylation site of the rat preproinsulin gene. Otherhigh efficiency promoters can also be used for the expression, e.g., thehuman β-actin promoter, the SV40 early or late promoters or the longterminal repeats from other retroviruses, e.g., HIV and HTLVI.Clontech's Tet-Off and Tet-On gene expression systems and similarsystems can be used to express the DR4 polypeptide in a regulated way inmammalian cells (Gossen, M., & Bujard, H. 1992, Proc. Natl. Acad. Sci.USA 89:5547–5551). For the polyadenylation of the mRNA other signals,e.g., from the human growth hormone or globin genes can be used as well.Stable cell lines carrying a gene of interest integrated into thechromosomes can also be selected upon co-transfection with a selectablemarker such as gpt, G418 or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g., G418 plusmethotrexate.

The plasmid pC4 is digested with the restriction enzyme BamHI and thendephosphorylated using calf intestinal phosphates by procedures known inthe art. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding the complete polypeptide is amplified usingPCR oligonucleotide primers corresponding to the 5′ and 3′ sequences ofthe desired portion of the gene. The 5′ primer containing the underlinedBamHI site, a Kozak sequence, and an AUG start codon, has the followingsequence:

-   5′, GCGGGATCCGCCATCATGGCGCCACCACCAGCTAGA 3′ (SEQ ID NO:9). The 3′    primer, containing the underlined BamHI site, has the following    sequence:-   5′, GCGGGATCCTCACTCCAAGGACACGGCAGAGCC 3′ (SEQ ID NO:10).

The amplified fragment is digested with the endonuclease BamHI and thenpurified again on a 1% agarose gel. The isolated fragment and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC4 using,for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene are used fortransfection. Five μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSVneo using lipofectin (Felgner et al.,supra). The plasmid pSV2-neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10–14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100–200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

EXAMPLE 3 Cloning and Expression of the Soluble Extracellular Domain ofDR4 in a Baculovirus Expression System

The cDNA sequence encoding the soluble extracellular domain of DR4protein in the deposited clone (ATCC No. 97853) is amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thegene:

-   The 5′ primer for DR4 has the sequence-   5′ GCGGGATCCGCCATCATGGCGCCACCACCAGCTAGA 3′ (SEQ ID NO:9) containing    the underlined BamHI restriction enzyme site. Inserted into an    expression vector, as described below, the 5′ end of the amplified    fragment encoding DR4 provides an efficient cleavage signal peptide.    An efficient signal for initiation of translation in eukaryotic    cells, as described by Kozak, M., J. Mol. Biol. 196:947–950 (1987)    is appropriately located in the vector portion of the construct.

The 3′ primer for both DR4 has the sequence 5′GCGGGATCCTCAATTATGTCCATTGCCTG 3′ (SEQ ID NO:11) containing theunderlined BamHI restriction followed by nucleotides complementary tothe DR4 nucleotide sequence set out in FIG. 1, followed by the stopcodon.

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean, ” BIO 101 Inc., La Jolla,Calif.) The fragment then is digested with BamHI and Asp718 and again ispurified on a 1% agarose gel.

The vector pA2 is used to express the DR4 protein in the baculovirusexpression system, using standard methods, such as those described inSummers et al., A Manual of Methods for Baculovirus Vectors and InsectCell Culture Procedures, Texas Agricultural Experimental StationBulletin No. 1555 (1987). This expression vector contains the strongpolyhedron promoter of the Autograph californica nuclear polyhedrosisvirus (ACMNPV) followed by convenient restriction sites. For an easyselection of recombinant virus the beta-galactosidase gene from E. coliis inserted in the same orientation as the polyhedron promoter and isfollowed by the polyadenylation signal of the polyhedron gene. Thepolyhedron sequences are flanked at both sides by viral sequences forcell-mediated homologous recombination with wild-type viral DNA togenerate viable virus that express the cloned polynucleotide.

Many other baculovirus vectors could be used in place of pA2, such aspAc373, pVL941 and pAcIM1 provided, as those of skill readily willappreciate, that construction provides appropriately located signals fortranscription, translation, trafficking and the like, such as anin-frame AUG and a signal peptide, as required. Such vectors aredescribed in Luckow et al., Virology 170:31–39, among others.

The plasmid is digested with the restriction enzyme Bam HI and then isdephosphorylated using calf intestinal phosphatase, using routineprocedures known in the art. The DNA is then isolated from a 1% agarosegel using a commercially available kit (“Geneclean” BIO 101 Inc., LaJolla, Calif.).

Fragment and the dephosphorylated plasmid are ligated together with T4DNA ligase. E. coli HB101 cells are transformed with ligation mix andspread on culture plates. Bacteria are identified that contain theplasmid with the human DDCR gene by digesting DNA from individualcolonies using BamHI and then analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing. This plasmid is designated herein pBac DR4.

5 μg of the plasmid pBac DR4 is co-transfected with 1.0 μg of acommercially available linearized baculovirus DNA (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al, Proc. Natl. Acad. Sci. USA84:7413–7417 (1987). 1 μg of BaculoGold™ virus DNA and 5 μg of theplasmid pBac DR4 are mixed in a sterile well of a microliter platecontaining 50 μl of serum free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is rocked back and forth tomix the newly added solution. The plate is then incubated for 5 hours at27 C. After 5 hours the transfection solution is removed from the plateand 1 ml of Grace's insect medium supplemented with 10% fetal calf serumis added. The plate is put back into an incubator and cultivation iscontinued at 27 C. for four days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, cited above. An agarosegel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used toallow easy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9–10).

Four days after serial dilution, the virus is added to the cells. Afterappropriate incubation, blue stained plaques are picked with the tip ofan Eppendorf pipette. The agar containing the recombinant viruses isthen resuspended in an Eppendorf tube containing 200 μl of Grace'smedium. The agar is removed by a brief centrifugation and thesupernatant containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatant of theseculture dishes are harvested and then they are stored at 4 C. A clonecontaining properly inserted DR4 is identified by DNA analysis includingrestriction mapping and sequencing. This is designated herein as V-DR4.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-DR4 at a multiplicity of infection (“MOI”) of about 2(about 1 to about 3). Six hours later the medium is removed and isreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Gaithersburg). 42 hours later, 5 gCi of³⁵S-methionine and 5 μCi ³⁵S cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then they areharvested by centrifugation, lysed and the labeled proteins arevisualized by SDS-PAGE and autoradiography.

EXAMPLE 4 Tissue Distribution of DR4 Gene Expression

Northern blot analysis is carried out to examine DR4 gene (ATCC No.97853) expression in human tissues, using methods described by, amongothers, Sambrook et al., cited above. A cDNA probe containing the entirenucleotide sequence of the DR4 protein (SEQ ID NO:1) is labeled with ³²Pusing the rediprime™ DNA labeling system (Amersham Life Science),according to manufacturer's instructions. After labeling, the probe ispurified using a CHROMA SPIN-100™ column (Clontech Laboratories, Inc.),according to manufacturer's protocol number PT1200-1. The purifiedlabeled probe is then used to examine various human tissues for DR4mRNA.

Multiple Tissue Northern (MTN) blots containing various human tissues(H) or human immune system tissues (IM) are obtained from Clontech andare examined with labeled probe using ExpressHyb™ hybridization solution(Clontech) according to manufacturer's protocol number PT1190-1.Following hybridization and washing, the blots are mounted and exposedto film at −70 C. overnight, and films developed according to standardprocedures Expression of DR4 was detected in tissues enriched inlymphocytes including amniotic cells, heart, liver cancer, kidney,peripheral blood leukocytes, activated T-cell, K562 plus PMA, W138cells, Th2 cells, human tonsils, and CD34 depleted buffy coat (cordblood). It can be envisaged that DR4 plays a role in lymphocytehomeostasis.

EXAMPLE 5 DR4 Induced Apoptosis

Overexpression of Fas/APO-1 and TNFR-1 in mammalian cells mimicsreceptor activation (M. Muzio, et al., Cell 85, 817–827 (1996); M. P.Boldin, et al., Cell 85, 803–815 (1996)). Thus, this system was utilizedto study the functional role of DR4. Transient expression of DR4 in MCF7human breast carcinoma cells and 293 human embryonic kidney cellsinduced rapid apoptosis.

Cell death assays are performed essentially as previously described (A.M. Chinnaiyan, et al., Cell 81, 505–12 (1995); M. P. Boldin, et al., JBiol Chem 270, 7795–8 (1995); F. C. Kischkel, et al., EMBO 14, 5579–5588(1995); A. M. Chinnaiyan, et al., J Biol Chem 271, 4961–4965 (1996)).Briefly, MCF-7 human breast carcinoma clonal cell lines stablytransfected with either vector alone or a CrmA expression construct (M.Tewari, et al., J Biol Chem 270, 3255–60 (1995)), are transientlytransfected with pCMV-DR4-galatosidase (or pCMV-DR4-galactosidase(lacking the death domain)) in the presence of a ten-fold excess ofpcDNA3 expression constructs encoding the indicated proteins usinglipofectamine (GIBCO-BRL). 293 cells are likewise transfected using theCaPO₄ method. The ICE family inhibitor z-VAD-fmk (Enzyme SystemsProducts, Dublin, Calif.) is added to the cells at a concentration of 10μM, 5 hrs after transfection. 32 hours following transfection, cells arefixed and stained with X-Gal as previously described (A. M. Chinnaiyan,et al., Cell 81, 505–12 (1995); M. P. Boldin, et al., J Biol Chem 270,7795–8 (1995); F. C. Kischkel, et al., EMBO 14, 5579–5588 (1995)).

The cells displayed morphological alterations typical of cellsundergoing apoptosis, becoming rounded, condensed and detaching from thedish. Similar to TNFR-1 and Fas/APO-1 (M. Muzio, et al., Cell 85,817–827 (1996); M. P. Boldin, et al., Cell 85, 803–815 (1996); M.Tewari, et al., J Biol Chem 270, 3255–60 (1995)), DR4-induced apoptosiswas blocked by the inhibitors of ICE-like proteases, CrmA and z-VAD-fmk

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosures of all patents, patent applications, andpublications referred to herein are hereby incorporated by reference.

1. A method of inducing apoptosis of a DR4-expressing cell, comprising contacting said cell with an agonist antibody or fragment thereof that specifically binds to a polypeptide consisting of amino acids 24 to 238 of SEQ ID NO:2.
 2. The method of claim 1 which is in vitro.
 3. The method of claim 1 which is in vivo.
 4. The method of claim 1, wherein the polypeptide is glycosylated.
 5. The method of claim 1, wherein said antibody or fragment thereof is polyclonal.
 6. The method of claim 1, wherein said antibody or fragment thereof is monoclonal.
 7. The method of claim 1, wherein said antibody or fragment thereof is selected from the group consisting of: (a) a chimeric antibody; (b) a Fab fragment; and (c) a F(ab′)₂ fragment.
 8. The method of claim 1, wherein said antibody or fragment thereof is labeled.
 9. The method of claim 8, wherein said label is selected from the group consisting of: (a) an enzyme; (b) a fluorescent label; and (c) a radioisotope.
 10. The method of claim 1, wherein said antibody or fragment thereof specifically binds to said polypeptide in a Western blot.
 11. The method of claim 1, wherein said antibody or fragment thereof specifically binds to said polypeptide in an ELISA.
 12. The method of claim 1, further comprising contacting said cell with a compound that potentiates apoptosis selected from the group consisting of: (a) TRAIL; and (b) a chemotherapeutic drug.
 13. The method of claim 12, wherein said compound is TRAIL.
 14. The method of claim 12, wherein said compound is a chemotherapeutic drug.
 15. A method of inducing apoptosis of a DR4-expressing cell, comprising contacting said cell with an agonist antibody or fragment thereof that specifically binds to a DR4 polypeptide expressed on the surface of a cell, wherein said polypeptide is encoded by a polynucleotide encoding amino acids 1–468 of SEQ ID NO:2.
 16. The method of claim 15 which is in vitro.
 17. The method of claim 15 which is in vivo.
 18. The method of claim 15, wherein the polypeptide is glycosylated.
 19. The method of claim 15, wherein said antibody or fragment thereof is polyclonal.
 20. The method of claim 15, wherein said antibody or fragment thereof is monoclonal.
 21. The method of claim 15, wherein said antibody or fragment thereof is selected from the group consisting of: (a) a chimenc antibody; (b) a Fab fragment; and (c) a F(ab′)₂ fragment.
 22. The method of claim 15, wherein said antibody or fragment thereof is labeled.
 23. The method of claim 22, wherein said label is selected from the group consisting of: (a) an enzyme; (b) a fluorescent label; and (c) a radioisotope.
 24. The method of claim 15, wherein said antibody or fragment thereof specifically binds to said polypeptide in a Western blot.
 25. The method of claim 15, wherein said antibody or fragment thereof specifically binds to said polypeptide in an ELISA.
 26. The method of claim 15, further comprising contacting said cell with a compound that potentiates apoptosis selected from the group consisting of: (a) TRAIL; and (b) a chemotherapeutic drug.
 27. The method of claim 26, wherein said compound is TRAIL.
 28. The method of claim 26, wherein said compound is a chemotherapeutic drug.
 29. A method of inducing apoptosis of a DR4-expressing cell, comprising contacting said cell with an agonist antibody or fragment thereof that specifically binds the extracellular domain of the polypeptide encoded by the cDNA clone contained in ATCC Deposit No.
 97853. 30. The method of claim 29 which is in vitro.
 31. The method of claim 29 which is in vivo.
 32. The method of claim 29, wherein the polypeptide is glycosylated.
 33. The method of claim 29, wherein said antibody or fragment thereof is polyclonal.
 34. The method of claim 29, wherein said antibody or fragment thereof is monoclonal.
 35. The method of claim 29, wherein said antibody or fragment thereof is selected from the group consisting of: (a) a chimeric antibody; (b) a Fab fragment; and (c) a F(ab′)₂ fragment.
 36. The method of claim 29, wherein said antibody or fragment thereof is labeled.
 37. The method of claim 36, wherein said label is selected from the group consisting of: (a) an enzyme; (b) a fluorescent label; and (c) a radioisotope.
 38. The method of claim 29, wherein said antibody or fragment thereof specifically binds to said polypeptide in a Western blot.
 39. The method of claim 29, wherein said antibody or fragment thereof specifically binds to said polypeptide in an ELISA.
 40. The method of claim 29, further comprising contacting said cell with a compound that potentiates apoptosis selected from the group consisting of: (a) TRAIL; and (b) a chemotherapeutic drug.
 41. The method of claim 40, wherein said compound is TRAIL.
 42. The method of claim 40, wherein said compound is a chemotherapeutic drug.
 43. A method of inducing apoptosis of a DR4-expressing cell comprising contacting said cell with an agonist antibody or fragment thereof that specifically binds to a DR4 polypeptide expressed on the surface of a cell, wherein said polypeptide is encoded by a polynucleotide encoding the polypeptide encoded by the cDNA contained in ATCC Deposit No.
 97853. 44. The method of claim 43 which is in vitro.
 45. The method of claim 43 which is in vivo.
 46. The method of claim 43, wherein the polypeptide is glycosylated.
 47. The method of claim 43, wherein said antibody or fragment thereof is polyclonal.
 48. The method of claim 43, wherein said antibody or fragment thereof is monoclonal.
 49. The method of claim 43, wherein said antibody or fragment thereof is selected from the group consisting of: (a) a chimeric antibody; (b) a Fab fragment; and (c) a F(ab′)₂ fragment.
 50. The method of claim 43, wherein said antibody or fragment thereof is labeled.
 51. The method of claim 50, wherein said label is selected from the group consisting of: (a) an enzyme; (b) a fluorescent label; and (c) a radioisotope.
 52. The method of claim 43, wherein said antibody or fragment thereof specifically binds to said polypeptide in a Western blot.
 53. The method of claim 43, wherein said antibody or fragment thereof specifically binds to said polypeptide in an ELISA.
 54. The method of claim 43, further comprising contacting said cell with a compound that potentiates apoptosis selected from the group consisting of: (a) TRAIL; and (b) a chemotherapeutic drug.
 55. The method of claim 54, wherein said compound is TRAIL.
 56. The method of claim 54, wherein said compound is a chemotherapeutic drug.
 57. A method of treating cancer, comprising administering to a patient an agonist antibody or fragment thereof that specifically binds to a polypeptide consisting of amino acids 24 to 238 of SEQ ID NO:2, wherein said antibody or fragment thereof is administered in an amount sufficient to induce apoptosis of a DR4-expressing cancer cell.
 58. The method of claim 57, wherein the polypeptide is glycosylated.
 59. The method of claim 57, wherein said antibody or fragment thereof is polyclonal.
 60. The method of claim 57, wherein said antibody or fragment thereof is monoclonal.
 61. The method of claim 57, wherein said antibody or fragment thereof is selected from the group consisting of: (a) a chimeric antibody; (b) a Fab fragment; and (c) a F(ab′)₂ fragment.
 62. The method of claim 57, wherein said antibody or fragment thereof is labeled.
 63. The method of claim 62, wherein said label is selected from the group consisting of: (a) an enzyme; (b) a fluorescent label; and (c) a radioisotope.
 64. The method of claim 57, wherein said antibody or fragment thereof specifically binds to said polypeptide in a Western blot.
 65. The method of claim 57, wherein said antibody or fragment thereof specifically binds to said polypeptide in an ELISA.
 66. The method of claim 57, further comprising administering to said patient a compound that potentiates apoptosis selected from the group consisting of: (a) TRAIL; and (b) a chemotherapeutic drug.
 67. The method of claim 66, wherein said compound is TRAIL.
 68. The method of claim 66, wherein said compound is a chemotherapeutic drug.
 69. A method of treating cancer, comprising administering to a patient an agonist antibody or fragment thereof that specifically binds to a DR4 polypeptide expressed on the surface of a cell, wherein said polypeptide is encoded by a polynucleotide encoding amino acids 1–468 of SEQ ID NO:2, and wherein said antibody or fragment thereof is administered in an amount sufficient to induce apoptosis of a DR4-expressing cancer cell.
 70. The method of claim 69, wherein the polypeptide is glycosylated.
 71. The method of claim 69, wherein said antibody or fragment thereof is polyclonal.
 72. The method of claim 69, wherein said antibody or fragment thereof is monoclonal.
 73. The method of claim 69, wherein said antibody or fragment thereof is selected from the group consisting of: (a) a chimeric antibody; (b) a Fab fragment; and (c) a F(ab′)₂ fragment.
 74. The method of claim 69, wherein said antibody or fragment thereof is labeled.
 75. The method of claim 74, wherein said label is selected from the group consisting of: (a) an enzyme; (b) a fluorescent label; and (c) a radioisotope.
 76. The method of claim 69, wherein said antibody or fragment thereof specifically binds to said polypeptide in a Western blot.
 77. The method of claim 69, wherein said antibody or fragment thereof specifically binds to said polypeptide in an ELISA.
 78. The method of claim 69, further comprising administering to said patient a compound that potentiates apoptosis selected from the group consisting of: (a) TRAIL; and (b) a chemotherapeutic drug.
 79. The method of claim 78, wherein said compound is TRAIL.
 80. The method of claim 78, wherein said compound is a chemotherapeutic drug.
 81. A method of treating cancer, comprising administering to a patient an agonist antibody or fragment thereof that specifically binds the extracellular domain of the polypeptide encoded by the cDNA clone contained in ATCC Deposit No. 97853, wherein said antibody or fragment thereof is administered in an amount sufficient to induce apoptosis of a DR4-expressing cancer cell.
 82. The method of claim 81, wherein the polypeptide is glycosylated.
 83. The method of claim 81, wherein said antibody or fragment thereof is polyclonal.
 84. The method of claim 81, wherein said antibody or fragment thereof is monoclonal.
 85. The method of claim 81, wherein said antibody or fragment thereof is selected from the group consisting of: (a) a chimeric antibody; (b) a Fab fragment; and (c) a F(ab′)₂ fragment.
 86. The method of claim 81, wherein said antibody or fragment thereof is labeled.
 87. The method of claim 86, wherein said label is selected from the group consisting of: (a) an enzyme; (b) a fluorescent label; and (c) a radioisotope.
 88. The method of claim 81, wherein said antibody or fragment thereof specifically binds to said polypeptide in a Western blot.
 89. The method of claim 81, wherein said antibody or fragment thereof specifically binds to said polypeptide in an ELISA.
 90. The method of claim 81, further comprising administering to said patient a compound that potentiates apoptosis selected from the group consisting of: (a) TRAIL; and (b) a chemotherapeutic drug.
 91. The method of claim 90, wherein said compound is TRAIL.
 92. The method of claim 90, wherein said compound is a chemotherapeutic drug.
 93. A method of treating cancer, comprising administering to a patient an agonist antibody or fragment thereof that specifically binds to DR4 polypeptide expressed on the surface of a cell, wherein said polypeptide is encoded by a polynucleotide encoding the polypeptide encoded by the cDNA contained in ATCC Deposit No. 97853, and wherein said antibody or fragment thereof is administered in an amount sufficient to induce apoptosis of a DR4-expressing cancer cell.
 94. The method of claim 93, wherein the polypeptide is glycosylated.
 95. The method of claim 93, wherein said antibody or fragment thereof is polyclonal.
 96. The method of claim 93, wherein said antibody or fragment thereof is monoclonal.
 97. The method of claim 93, wherein said antibody or fragment thereof is selected from the group consisting of: (a) a chimeric antibody; (b) a Fab fragment; and (c) a F(ab′)₂ fragment.
 98. The method of claim 93, wherein said antibody or fragment thereof is labeled.
 99. The method of claim 98, wherein said label is selected from the group consisting of: (a) an enzyme; (b) a fluorescent label; and (c) a radioisotope.
 100. The method of claim 93, wherein said antibody or fragment thereof specifically binds to said polypeptide in a Western blot.
 101. The method of claim 93, wherein said antibody or fragment thereof specifically binds to said polypeptide in an ELISA.
 102. The method of claim 93, further comprising administering to said patient a compound that potentiates apoptosis selected from the group consisting of: (a) TRAIL; and (b) a chemotherapeutic drug.
 103. The method of claim 102, wherein said compound is TRAIL.
 104. The method of claim 102, wherein said compound is a chemotherapeutic drug.
 105. A method of inducing apoptosis of a DR4-expressing cell, comprising contacting said cell with an antibody or fragment thereof that specifically binds to a polypeptide consisting of amino acids 24 to 238 of SEQ ID NO:2, wherein said antibody or fragment thereof induces apoptosis in a DR4-expressing cell.
 106. The method of claim 105 which is in vitro.
 107. The method of claim 105 which is in vivo.
 108. The method of claim 105, wherein the polypeptide is glycosylated.
 109. The method of claim 105, wherein said antibody or fragment thereof is polyclonal.
 110. The method of claim 105, wherein said antibody or fragment thereof is monoclonal.
 111. The method of claim 105, wherein said antibody or fragment thereof is selected from the group consisting of: (a) a chimeric antibody; (b) a Fab fragment; and (c) a F(ab′)₂ fragment.
 112. The method of claim 105, wherein said antibody or fragment thereof is labeled.
 113. The method of claim 112, wherein said label is selected from the group consisting of: (a) an enzyme; (b) a fluorescent label; and (c) a radioisotope.
 114. The method of claim 105, wherein said antibody or fragment thereof specifically binds to said polypeptide in a Western blot.
 115. The method of claim 105, wherein said antibody or fragment thereof specifically binds to said polypeptide in an ELISA.
 116. The method of claim 105, further comprising contacting said cell with a compound that potentiates apoptosis selected from the group consisting of: (a) TRAIL; and (b) a chemotherapeutic drug.
 117. The method of claim 116, wherein said compound is TRAIL.
 118. The method of claim 116, wherein said compound is a chemotherapeutic drug.
 119. A method of inducing apoptosis of a DR4-expressing cell, comprising contacting said cell with an antibody or fragment thereof that specifically binds to a DR4 polypeptide expressed on the surface of a cell, wherein said polypeptide is encoded by a polynucleotide encoding amino acids 1–468 of SEQ ID NO:2, and wherein said antibody or fragment thereof induces apoptosis in a DR4-expressing cell.
 120. The method of claim 119 which is in vitro.
 121. The method of claim 119 which is in vivo.
 122. The method of claim 119, wherein the polypeptide is glycosylated.
 123. The method of claim 119, wherein said antibody or fragment thereof is polyclonal.
 124. The method of claim 119, wherein said antibody or fragment thereof is monoclonal.
 125. The method of claim 119, wherein said antibody or fragment thereof is selected from the group consisting of: (a) a chimeric antibody; (b) a Fab fragment; and (c) a F(ab′)₂ fragment.
 126. The method of claim 119, wherein said antibody or fragment thereof is labeled.
 127. The method of claim 126, wherein said label is selected from the group consisting of: (a) an enzyme; (b) a fluorescent label; and (c) a radioisotope.
 128. The method of claim 119, wherein said antibody or fragment thereof specifically binds to said polypeptide in a Western blot.
 129. The method of claim 119, wherein said antibody or fragment thereof specifically binds to said polypeptide in an ELISA.
 130. The method of claim 119, further comprising contacting said cell with a compound that potentiates apoptosis selected from the group consisting of: (a) TRAIL; and (b) a chemotherapeutic drug.
 131. The method of claim 130, wherein said compound is TRAIL.
 132. The method of claim 130, wherein said compound is a chemotherapeutic drug.
 133. A method of inducing apoptosis of a DR4-expressing cell, comprising contacting said cell with an antibody or fragment thereof that specifically binds the extracellular domain of the polypeptide encoded by the cDNA clone contained in ATCC Deposit No. 97853, wherein said antibody or fragment thereof induces apoptosis in a DR4-expressing cell.
 134. The method of claim 133 which is in vitro.
 135. The method of claim 133 which is in vivo.
 136. The method of claim 133, wherein the polypeptide is glycosylated.
 137. The method of claim 133, wherein said antibody or fragment thereof is polyclonal.
 138. The method of claim 133, wherein said antibody or fragment thereof is monoclonal.
 139. The method of claim 133, wherein said antibody or fragment thereof is selected from the group consisting of: (a) a chimeric antibody; (b) a Fab fragment; and (c) a F(ab′)₂ fragment.
 140. The method of claim 133, wherein said antibody or fragment thereof is labeled.
 141. The method of claim 140, wherein said label is selected from the group consisting of: (a) an enzyme; (b) a fluorescent label; and (c) a radioisotope.
 142. The method of claim 133, wherein said antibody or fragment thereof specifically binds to said polypeptide in a Western blot.
 143. The method of claim 133, wherein said antibody or fragment thereof specifically binds to said polypeptide in an ELISA.
 144. The method of claim 133, further comprising contacting said cell with a compound that potentiates apoptosis selected from the group consisting of: (a) TRAIL; and (b) a chemotherapeutic drug.
 145. The method of claim 144, wherein said compound is TRAIL.
 146. The method of claim 144, wherein said compound is a chemotherapeutic drug.
 147. A method of inducing apoptosis of a DR4-expressing cell, comprising contacting said cell with an antibody or fragment thereof that specifically binds to a DR4 polypeptide expressed on the surface of a cell, wherein said polypeptide is encoded by a polynucleotide encoding the polypeptide encoded by the cDNA clone contained in ATCC Deposit No. 97853, and wherein said antibody or fragment thereof induces apoptosis in a DR4-expressing cell.
 148. The method of claim 147 which is in vitro.
 149. The method of claim 147, which is in vivo.
 150. The method of claim 147, wherein the polypeptide is glycosylated.
 151. The method of claim 147, wherein said antibody or fragment thereof is polyclonal.
 152. The method of claim 147, wherein said antibody or fragment thereof is monoclonal.
 153. The method of claim 147, wherein said antibody or fragment thereof is selected from the group consisting of: (a) a chimeric antibody; (b) a Fab fragment; and (c) a F(ab′)₂ fragment.
 154. The method of claim 147, wherein said antibody or fragment thereof is labeled.
 155. The method of claim 154, wherein said label is selected from the group consisting of: (a) an enzyme; (b) a fluorescent label; and (c) a radioisotope.
 156. The method of claim 147, wherein said antibody or fragment thereof specifically binds to said polypeptide in a Western blot.
 157. The method of claim 147, wherein said antibody or fragment thereof specifically binds to said polypeptide in an ELISA.
 158. The method of claim 147, further comprising contact said cell with a compound that potentiates opoptosis selected from the group consisting of (a) TRAIL; and (b) a chemotherapy drug.
 159. The method of claim 158, wherein said compound is TRAIL.
 160. The method of claim 158, wherein said compound is a chemotherapeutic drug.
 161. A method of treating cancer, comprising administering to a patient an antibody or fragment thereof that specifically binds to a polypeptide consisting of amino acids 24 to 238 of SEQ ID NO:2, wherein said antibody or fragment thereof is administered in an amount sufficient to induce apoptosis of a DR4-expressing cancer cell.
 162. The method of claim 161, wherein the polypeptide is glycosylated.
 163. The method of claim 161, wherein said antibody or fragment thereof is polyclonal.
 164. The method of claim 161, wherein said antibody or fragment thereof is monoclonal.
 165. The method of claim 161, wherein said antibody or fragment thereof is selected from the group consisting of: (a) a chimeric antibody; (b) a Fab fragment; and (c) a F(ab′)₂ fragment.
 166. The method of claim 161, wherein said antibody or fragment thereof is labeled.
 167. The method of claim 166, wherein said label is selected from the group consisting of: (a) an enzyme; (b) a fluorescent label; and (c) a radioisotope.
 168. The method of claim 161, wherein said antibody or fragment thereof specifically binds to said polypeptide in a Western blot.
 169. The method of claim 161, wherein said antibody or fragment thereof specifically binds to said polypeptide in an ELISA.
 170. The method of claim 161, further comprising administering to said patient a compound that potentiates apoptosis selected from the group consisting of: (a) TRAIL; and (b) a chemotherapeutic drug.
 171. The method of claim 170, wherein said compound is TRAIL.
 172. The method of claim 170, wherein said compound is a chemotherapeutic drug.
 173. A method of treating cancer, comprising administering to a patient an antibody or fragment thereof that specifically binds to a DR4 polypeptide expressed on the surface of a cell, wherein said polypeptide is encoded by a polynucleotide encoding amino acids 1–468 of SEQ ID NO:2, wherein said antibody or fraament thereof is administered in an amount sufficient to induce apoptosis of a DR4-expressing cancer cell.
 174. The method of claim 173, wherein the polypeptide is glycosylated.
 175. The method of claim 173, wherein said antibody or fragment thereof is polyclonal.
 176. The method of claim 173, wherein said antibody or fragment thereof is monoclonal.
 177. The method of claim 173, wherein said antibody or fragment thereof is selected from the group consisting of: (a) a chimeric antibody; (b) a Fab fragment; and (c) a F(ab′)₂ fragment.
 178. The method of claim 173, wherein said antibody or fragment thereof is labeled.
 179. The method of claim 178, wherein said label is selected from the group consisting of: (a) an enzyme; (b) a fluorescent label; and (c) a radioisotope.
 180. The method of claim 173, wherein said antibody or fragment thereof specifically binds to said polypeptide in a Western blot.
 181. The method of claim 173, wherein said antibody or fragment thereof specifically binds to said polypeptide in an ELISA.
 182. The method of claim 173, further comprising administering to said patient a compound that potentiates apoptosis selected from the group consisting of: (a) TRAIL; and (b) a chemotherapeutic drug.
 183. The method of claim 182, wherein said compound is TRAIL.
 184. The method of claim 182, wherein said compound is a chemotherapeutic drug.
 185. A method of treating cancer, comprising administering to a patient an antibody or fragment thereof that specifically binds the extracellular domain of the polypeptide encoded by the cDNA clone contained in ATCC Deposit No. 97853, wherein said antibody or fragment thereof is administered in an amount sufficient to induce apoptosis of a DR4-expressing cancer cell.
 186. The method of claim 185, wherein the polypeptide is glycosylated.
 187. The method of claim 185, wherein said antibody or fragment thereof is polyclonal.
 188. The method of claim 185, wherein said antibody or fragment thereof is monoclonal.
 189. The method of claim 185, wherein said antibody or fragment thereof is selected from the group consisting of: (a) a chimeric antibody; (b) a Fab fragment; and (c) a F(ab′)₂ fragment.
 190. The method of claim 185, wherein said antibody or fragment thereof is labeled.
 191. The method of claim 190, wherein said label is selected from the group consisting of: (a) an enzyme; (b) a fluorescent label; and (c) a radioisotope.
 192. The method of claim 185, wherein said antibody or fragment thereof specifically binds to said polypeptide in a Western blot.
 193. The method of claim 185, wherein said antibody or fragment thereof specifically binds to said polyp eptide in an ELISA.
 194. The method of claim 185, further comprising administering to said patient a comtound that potentiates apoptosis selected from the group consisting of: (a) TRAIL; and (b) a chemotherapeutic drug.
 195. The method of claim 194, wherein said compound is TRAIL.
 196. The method of claim 194, wherein said compound is a chemotherapeutic drug.
 197. A method of treating cancer, comprising administering to a patient an antibody or fragment thereof that specifically binds to a DR4 polypeptide expressed on the surface of a cell, wherein said polypeptide is encoded by a polynucleotide encoding the polypeptide encoded by the cDNA clone contained in ATCC Deposit No. 97853, wherein said antibody or fragment thereof is administered in an amount sufficient to induce apoptosis of a DR4-expressing cancer cell.
 198. The method of claim 197, wherein the polypeptide is glycosylated.
 199. The method of claim 197, wherein said antibody or fragment thereof is polyclonal.
 200. The method of claim 197, wherein said antibody or fragment thereof is monoclonal.
 201. The method of claim 197, wherein said antibody or fragment thereof is selected from the group consisting of: (a) a chimeric antibody; (b) a Fab fragment; and (c) a F(ab′)₂ fragment.
 202. The method of claim 197, wherein said antibody or fragment thereof is labeled.
 203. The method of claim 202, wherein said label is selected from the group consisting of: (a) an enzyme; (b) a fluorescent label; and (c) a radioisotope.
 204. The method of claim 197, wherein said antibody or fragment thereof specifically binds to said polypeptide in a Western blot.
 205. The method of claim 197, wherein said antibody or fragment thereof specifically binds to said polypeptide in an ELISA.
 206. The method of claim 197, further comprising administering to said patient a compound that potentiates apoptosis selected from the group consisting of: (a) TRAIL; and (b) a chemotherapeutic drug.
 207. The method of claim 206, wherein said compound is TRAIL.
 208. The method of claim 206, wherein said compound is a chemotherapeutic drug.
 209. A composition comprising (i) an agonist antibody or fragment thereof that specifically binds to a polypeptide consisting of amino acids 24 to 238 of SEQ ID NO:2, and (ii) a compound that potentiates apoptosis selected from the group consisting of: (a) TRAIL, and (b) a chemotherapeutic drug.
 210. The composition of claim 209, wherein said compound is TRAIL.
 211. The composition of claim 209, wherein said compound is a chemotherapeutic drug.
 212. A composition comprising (i) an agonist antibody or fragment thereof that specifically binds to a DR4 polypeptide expressed on the surface of a cell, wherein said polypeptide is encoded by a polynucleotide encoding amino acids 1–468 of SEQ ID NO:2, and (ii) a compound that potentiates apoptosis selected from the group consisting of: (a) TRAIL, and (b) a chemotherapeutic drug.
 213. The composition of claim 212, wherein said compound is TRAIL.
 214. The composition of claim 212, wherein said compound is a chemotherapeutic drug.
 215. A composition comprising (i) an agonist antibody or fragment thereof that specifically binds the extracellular domain of the polypeptide encoded by the cDNA clone contained in ATCC Deposit No. 97853, and (ii) a compound that potentiates apoptosis selected from the group consisting of: (a) TRAIL, and (b) a chemotherapeutic drug.
 216. The composition of claim 215, wherein said compound is TRAIL.
 217. The composition of claim 215, wherein said compound is a chemotherapeutic drug.
 218. A composition comprising (i) an agonist antibody or fragment thereof that specifically binds to a DR4 polypeptide expressed on the surface of a cell, wherein said polypeptide is encoded by a polynucleotide encoding the polypeptide encoded by the cDNA contained in ATCC Deposit No. 97853, and (ii) a compound that potentiates apoptosis selected from the group consisting of: (a) TRAIL, and (b) a chemotherapeutic drug.
 219. The composition of claim 218, wherein said compound is TRAIL.
 220. The composition of claim 218, wherein said compound is a chemotherapeutic drug. 